Method for preventing the activation of inactive, recombinant Helicobacter pylori apourease

ABSTRACT

A method for preventing the activation of inactive, recombinant Helicobacter pylori apourease is presented. The method comprises contacting the apourease with a compound that modifies a sulfhydryl or lysyl residue of the urease. The covalently modified apourease is prevented from molecular aggregation.

BACKGROUND OF THE INVENTION

This invention relates to chemical and genetic methods for stabilizingHelicobacter urease.

Helicobacter pylori is a gram-negative bacterium and a gastroduodenalpathogen that causes gastritis, gastric and duodenal ulceration, andpossibly gastric carcinoma in humans (Graham et al., Am. J.Gastroenterol. 82:283-286, 1987; Homick, N. Eng. J. Med. 316:1598-1600,1987; Lee et al., Microb. Ecol. Health Dis. 1:1-16, 1988; Cover et al.,Annu. Rev. Med. 40:269-285, 1989; Buck, Clin. Microbiol. Rev. 3:1-12,1990). The bacterium produces large amounts of the enzyme urease, whichis a multimeric nickel metallohydrolase that cleaves urea to ammonia andcarbon dioxide (Hu et al., Infect. Immun. 58:992-998, 1990). H. pyloriurease is localized both in the cytosol and on the extracellular surfaceof the bacterium (Hawtin et al., J. Gen. Microbiol. 136:1995-2000,1990). Extracellular urease may protect the bacteria in the highlyacidic stomach by hydrolyzing urea to ammonia, thereby creating abuffering cloud of ammonia that neutralizes the acid around the bacteria(Ferrero et al., Microbiol. Ecol. Health Dis. 4:121-134, 1991; Mobley etal., In Helicobacter pylori: Basic Mechanisms to Clinical Cure, Hunt etal. (Eds.) Kluver Acad. Pub., Dordrecht, 1994). Urease may alsocontribute to the pathogenicity of H. pylori by direct toxicity ofammonia and monochloramine to cells lining the gastric mucosa.

H. pylori urease is immunogenic to humans and antigenicity is highlyconserved among H. pylori strains (Ferrero et al., Mol. Microbiol9:323-333, 1993; Gootz et al., Infect. Immun. 62:793-798, 1994).Antigenic conservation among ureases is the basis of protection of miceagainst H. felis infection when vaccinated with H. pylori urease(Ferrero et al., Mol. Microbiol 9:323-333, 1993). Antigeniccross-reactivity has also been demonstrated between H. pylori and H.mustelae ureases (Gootz et al., Infect. Immun. 62:793-798, 1994).

Urease enzymatic activity is toxic to animals and humans (Thomson etal., Am. J. Med. 35:804-812, 1963; Mobley et al., Microbiol. Rev.53:85-108, 1985; LeVeen et al., Biomed. Pharmacother. 48(3-4):157-166,1994). Anti-urease antibodies bind to urease, but generally do notinhibit urease enzymatic activity. For example, there is one report thatclaims to show inhibition of urease activity by monoclonal antibodies tourease (Nagata et al., Infect. Immun. 60:4826-4831, 1992). We testedmonoclonal and polyclonal antibodies to urease and urease subunits, andfound no inhibition of urease activity. Thomas et al. (J. Clin.Microbiol. 30:1338-1340) measured urease inhibitory activity in serumsamples from children infected with H. pylori, and found that amongthirteen serum samples showing urease binding activity, only one sampleshowed any urease inhibitory activity. These observations show that thecatalytic and immunogenic domains of urease are different. This isfurther supported by a recent report that antigenic reactivity of aurease preparation was retained under storage conditions in whichenzymatic activity was lost (Perez-Perez, Infect. Immun. 62:299-302,1994). These results suggest that immunogenicity can be separated frompotentially toxic enzymatic activity, which is an importantcharacteristic of a potential vaccine.

Specific human antibody responses to urease are absent or weak in a highproportion, if not the majority, of infected individuals. Antibodies tourease administered to animals together with live Helicobacter protectagainst infection (Blanchard et al., Infect. Immun. 63:1394-1395, 1995).Together, these observations support the basis for the effectiveness ofurease as a vaccine that induces a high-grade, urease-specific immuneresponse protective against H. pylori.

Nine genes have been identified in the H. pylori urease gene cluster(Cussac et al., J. Bacteriol. 174:2466-2473, 1992; Labigne et al., J.Bacteriol. 173:1920-193, 1992). These include the urease structuralgenes, encoding UreA and UreB, and the accessory genes, encoding Ure I,E, F, G, and H. These genes have been shown to be essential for ureaseactivity. Hu et al. (Infect. Immun. 60:2657-2666, 1992) expressed genesencoding the UreA and UreB subunits of H. pylori urease in E. coli andshowed that these two genes alone are sufficient to encode a fullyassembled apoenzyme, which was structurally and immunologicallyidentical to native urease, but catalytically inactive, due to theabsence of nickel ions. Addition of nickel ions alone did not restorecatalytic activity. Similar results were reported with other bacterialureases. In other studies, recombinant Klebsiella aerogenes apourease,which differs substantially from H. pylori urease in subunit structureand overall amino acid sequence, was shown to be activated in vitro byincubation with carbon dioxide or bicarbonate, together with nickel ions(Park et al., Science 267:1156-1158, 1995). Based on crystallographicanalysis, the K. aerogenes urease bi-nickel center is thought to includeHis 272, His 246, His 136, His 134, Asp 360, and Lys 219 of the Ure Csubunit. By analogy, the H. pylori urease metallocenter may be describedas including His 248, His 138, His 136, Asp 362, and Lys 219 of the UreB subunit.

SUMMARY OF THE INVENTION

We have shown that treatment of Helicobacter urease with compounds thatchemically modify urease amino acids can prevent in vitro activation ofurease, as well as molecular aggregation of urease. We have also shownthat including amino acid substitutions in urease can prevent in vitroactivation of urease.

Accordingly, in one aspect, the invention features methods ofstabilizing Helicobacter (e.g., H. pylori) urease. In these methods,urease (e.g., recombinant or native urease) is contacted with a compoundthat modifies an amino acid of the urease. Stabilization of ureaseaccording to the invention can prevent activation of the urease, orprevent molecular aggregation of the urease.

Compounds used in these methods can be, for example, sulfhydryl-reactivecompounds. For example, iodoacetamide (IAM), iodoacetic acid (IAA),5-5'-dithio-bis-2-nitrobenzoic acid (DTNB), 2,2'-dithiodipyridine(DTDP), cystine, cystamine, methyl methanethiolsulfonate (MMTS), N-ethylmaleimide (NEM), dinitrofluorobenzene (DNFB), or trinitrobenzenesulfonic acid (TNBSA) can be used. Additional compounds that can be usedin the invention are described further below.

Also included in the invention are Helicobacter (e.g., H. pylori) ureasepolypeptides, for example, recombinant or native urease polypeptides,that have been stabilized by any of the methods described above. Thestabilized urease polypeptides can be present in a pharmaceuticallyacceptable carrier or diluent.

In another aspect, the invention provides Helicobacter (e.g., H. pylori)urease polypeptides having an amino acid mutation (or mutations) thatprevents activation of the urease polypeptides. For example, the ureasepolypeptides can contain an amino acid mutation in urease amino acidhistidine 136, histidine 248, or lysine 219 in UreB. The ureasepolypeptides having such a mutation can be present in a pharmaceuticallyacceptable carrier or diluent.

Also included in the invention are methods of inducing an immuneresponse to Helicobacter in a patient. In these methods, one of theurease polypeptides described above is administered to a patient that isat risk of developing, but does not have, Helicobacter infection, or toa patient that has Helicobacter infection. The urease polypeptide can beadministered to a mucosal surface of the patient, or it can beadministered parenterally, e.g., by intravenous, intramuscular, orpercutaneous administration.

As used herein, a polypeptide, such as a Helicobacter ureasepolypeptide, is said to be "stabilized" if it has been treated ormodified so that it maintains its molecular structure, to a degreesufficient to retain immunogenicity. Urease that has been treated with acompound, e.g., iodoacetamide (IAM), 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB), N-ethyl maleimide (NEM), or dinitrofluorobenzene (DNFB), so thatit is prevented from the molecular aggregation or activation (e.g., invitro activation) characteristic of untreated urease (see below) is saidto be "stabilized." The term "urease," as used herein, includes ureasepolypeptides that have been purified from an organism, such as abacterium from the genus Helicobacter (e.g., a bacterium of the speciesH. pylori), fragments of purified urease, as well as urease polypeptides(e.g., urease apoenzyme), urease subunits (e.g., UreA and UreBsubunits), and urease fragments produced using recombinant or chemicalsynthetic methods.

The invention provides several advantages. For example, maintenance ofurease in a stable molecular form can contribute to consistency in theresults of methods employing urease, such as prophylactic, therapeutic,and diagnostic methods. In addition, because activated urease has beenshown to be toxic in cell culture assays, animals, and humans (Thomsonet al., Am. J. Med. 35:804-812, 1963; Mobley et aL, Microbiol. Rev.53:85-108, 1985; LeVeen et aL, Biomed. Pharmacother. 48(3-4):157-166,1994) prevention of urease activation enables production of a saferurease product for use in prophylactic and therapeutic methods.

Other features and advantages of the invention will be apparent from thedetailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the activation of recombinant H. pylori ureaseby nickel ions and bicarbonate. Urease that was freeze-dried in 2%sucrose was reconstituted in water to a concentration of 4.0 mg/ml in 2%sucrose. Reconstituted urease was mixed with an equal volume of HEPES(0.1 M), NaCl (0.3 M), EDTA (1 mM) buffer, with or without sodiumbicarbonate and/or Ni⁺⁺ ions. The sample (1) incubation mixturecontained 0.2 M sodium bicarbonate and 200 μM Ni⁺⁺, the sample (2)incubation mixture contained 0.2 M sodium bicarbonate and no Ni⁺⁺, andthe sample (3) incubation mixture contained 200 μM Ni⁺⁺ and nobicarbonate. All of the samples were incubated for 40 minutes at 37° C.and were then assayed for urease activity using the urease-broth assay.Twenty μl of each sample were added to 1 ml of urease broth, mixed well,and immediately scanned at 550 nm to monitor the pH-dependentdeprotonation of phenol red to produce pink colored phenolate anion.Time course scanning was carried out in a Shimadzu model UV-1 PCspectrophotometer using a CPM 260 multi-channel cuvette holder. Thescans were normalized for zero time correction.

FIG. 2A is a photograph showing the activation of recombinant H. pyloriurease by Ni⁺⁺ ions and bicarbonate. Recombinant urease was incubatedwith HEPES, NaCl, EDTA buffer containing bicarbonate and/or Ni⁺⁺ ions,as described above for FIG. 1. Samples 1 and 2 were incubated with bothbicarbonate and Ni⁺⁺, samples 3 and 4 were incubated with bicarbonatealone, and samples 5 and 6 were incubated with Ni⁺⁺ alone. After 35minutes of incubation at 37° C., the samples were transferred to a 4° C.refrigerator and stored overnight. The samples were then fractionated ona 4% polyacrylamide gel (precast gel from Novex) under non-reducing andnon-dissociating conditions and stained using Coomassie blue.

FIG. 2B is a photograph of urease-specific silver staining of thefractionated samples described above for FIG. 2A. (See, e.g., de Llanoet al., Analytical Biochemistry 77:37-40, 1989, for details ofurease-specific silver staining protocol.)

FIG. 3 is a graph showing the kinetics of activation of recombinant H.pylori urease incubated with Ni⁺⁺ ions and bicarbonate. Recombinant H.pylori urease was incubated at 37° C. with activation buffer. Theactivation mixture contained 2 mg/ml urease, 100 mM HEPES (pH 8.3), 0.3M NaCl, 1 mM EDTA, 200 μM Ni⁺⁺, and 200 mM sodium bicarbonate. At theindicated time points, 10 μl of the activation mixture was transferredto 1 ml urea-broth, mixed well, and the absorbance at 550 nm followedfor 15 minutes. The zero time reading is subtracted from the 15 minutereading and plotted against the time of activation at 37° C. in theactivation system (1). Curves 2-4 show the results of Jack bean ureaseassayed at the different time points. The concentrations of urease inthe assay system are (1) 20 μg/ml activated, recombinant H. pyloriurease, (2) 2 μg/ml Jack bean urease, (3) 1 μg/ml Jack bean urease, and(4) 0.5 μg/ml Jack bean urease.

FIG. 4 is a graph showing that activation of recombinant H. pyloriapourease increases with increasing Ni⁺⁺ ion concentrations (2 μM, 20μM, 200 μM, and 1 mM). Recombinant H. pylori apourease was incubated at37° C. for 1 hour in activation buffer (100 mM HEPES (pH 8.3), 0.3 MNaCl, 1 mM EDTA, 200 mM sodium bicarbonate) containing different amountsof Ni⁺⁺, as indicated in the Figure. After 1 hour of incubation, 10 μlof activated urease was transferred to 1 ml of urea broth, mixed well,and increases in absorbance at 550 nm were monitored for 15 minutes.

FIG. 5 is a graph showing that the activation of recombinant H. pyloriapourease increases with increasing bicarbonate concentrations.Recombinant H. pylori apourease was incubated at 37° C. for 1 hour inactivation buffer (100 mM HEPES (pH 8.3), 0.3 M NaCl, 1 mM EDTA, 200 μMNi⁺⁺) containing different amounts of sodium bicarbonate, as indicatedin the Figure. After 1 hour of incubation, 10 μl of activated urease wastransferred to 1 ml of urea broth, mixed well, and increases inabsorbance at 550 nm were monitored for 15 minutes.

FIG. 6 is a photograph of a gel showing the effect of Ni⁺⁺ ions andbicarbonate on the activation of recombinant urease. Recombinant H.pylori urease was incubated at 37° C. for 1 hour in activation buffercontaining 200 mM bicarbonate and different concentrations of Ni⁺⁺ (lane1 is 2 μM Ni⁺⁺, lane 2 is 20 μM Ni⁺⁺, lane 3 is 200 μM Ni⁺⁺, and lane 4is 1 mM Ni⁺⁺), 200 μM Ni⁺⁺ with 5 mM bicarbonate (lane 5), or 200 μMNi⁺⁺ with 200 mM bicarbonate (lane 6). Samples were mixed with an equalvolume of non-reducing and non-associating sample buffer andfractionated by electrophoresis on a 4% native polyacrylamide gel. Afterelectrophoresis, the gel was stained using the urease activity-specificsilver staining protocol. Lane 7 contains jack bean urease.

FIG. 7A is a graph showing that activation of recombinant H. pyloriurease with bicarbonate and nickel ions is temperature dependent.Recombinant H. pylori apourease (2.0 mg/ml) was incubated in 100 mMHEPES (pH 8.3), 0.3 M NaCl, 1 mM EDTA, 200 μM Ni⁺⁺, 200 mM sodiumbicarbonate for 1 hour at the indicated temperatures (temperature isexpressed in absolute degrees (° K.)). After 1 hour of incubation, 10 μlof the activated urease was transferred to 1 ml of urease broth, mixedwell, and the increase in absorbance was then monitored at 550 nmn.

FIG. 7B is an Arrhenius plot of urease activation, which was constructedusing relative activity, as measured by increase in absorbance (FIG. 7A)and absolute temperature.

FIG. 8 is a graph showing that in vitro activation of recombinant H.pylori urease by Ni⁺⁺ and bicarbonate is pH dependent. Recombinant H.pylori apourease was incubated with 100 mM HEPES buffer, at theindicated pHs, containing 200 mM bicarbonate, 200 μM Ni⁺⁺ ions, 1 mMEDTA, and 0.3 M NaCl for 1 hour at 37° C. After 1 hour of incubation, 10μl of the urease sample was added to 1 ml urea broth, and the increasein absorbance at 550 nm was monitored.

FIG. 9 is a graph showing that activated recombinant H. pylori ureaseretains catalytic activity after dialysis of unbound nickel andbicarbonate. Recombinant H. pylori apourease was incubated for 1 hour at37° C. with 0.1 M HEPES (pH 8.3), 0.3 M NaCl, 1 mM EDTA, 1 mM Ni⁺⁺, and200 mM sodium bicarbonate. Samples without bicarbonate or Ni⁺⁺ were alsoincubated at the same time. After 1 hour of incubation, the samples weredialyzed against 2% sucrose overnight. The dialyzed samples wereanalyzed immediately after dialysis and also after 6 days of storage at4° C. after dialysis. Only urease that was preincubated with bothbicarbonate and Ni⁺⁺ showed activity. Activity was retained after 6 daysof storage at 4° C. in 2% sucrose. The curves are labeled as follows:urease incubated with Ni⁺⁺ plus bicarbonate immediately after dialysis(a) and 6 days after dialysis (b); urease incubated with Ni⁺⁺immediately after dialysis (c) and 6 days after dialysis (d); ureaseincubated with bicarbonate immediately after dialysis (e) and 6 daysafter dialysis (f).

FIG. 10 is a graph showing a comparison of the affinities of activatedH. pylori urease and jack bean urease for urea. Recombinant H. pyloriurease was activated in vitro by incubation in urease activation bufferfor 1 hour at 37° C. The activation system contained 2 mg/ml urease, 100mM HEPES, 1 mM EDTA, 0.3 M NaCl, 200 mM sodium bicarbonate, and 1 mMNi⁺⁺. After 1 hour of incubation at 37° C., the activated urease wasdialyzed overnight against 2% sucrose. The activity of the urease wasdetermined over urea concentrations ranging from 0.2-10.0 mM using theurease-coupled glutamate dehydrogenase assay. The assay system contained50 mM Tris-HCl (pH 7.5), 1 mg/ml α-ketoglutarate, 0.4 mg/ml NADH,6.5-300 μg/ml glutamate dehydrogenase, and varying concentrations ofurea. Activated H. pylori urease and jack bean urease were used assources of urease enzyme. The reaction tubes were blanked without ureaseand NADH. NADH was added to all of the tubes and absorbance wasmonitored for 1-2 minutes. One hundred and ten μl of urease enzyme wasadded, rapidly mixed, and decreases in absorbance at 340 nm weremonitored. The slope of the best linear fit region of the kinetics of340 nm absorbance decrease was taken as a measure of the initialvelocity. A Lineweaver-Burk plot (1/V vs 1/S) was constructed and aK_(m) value was determined from the slope and intercept.

FIG. 11 is a graph showing the effect of other metal ions andbicarbonate on recombinant H. pylori apourease. Recombinant H. pyloriapourease was incubated with 0.1 M HEPES (pH 8.3), 0.5 M sodiumchloride, 1 mM EDTA, 200 mM sodium bicarbonate, and 200 μM of differentmetallic ions, as indicated in the Figure, for 1 hour at 37° C. After 1hour of incubation, 10 μl of the urease sample were added to 1 ml ofurea broth, and the absorbance at 550 nm was monitored.

FIG. 12 is a graph showing that manganous and bicarbonate-activatedrecombinant H. pylori urease lost catalytic activity after dialysis.Recombinant H. pylori apourease was activated by incubation with 100 mMHEPES (pH 8.0), 0.3 M NaCl, 1 mM EDTA, 200 mM sodium bicarbonate, andeither 1 mM Ni⁺⁺ (a and c) or 1 mM Manganous Chloride (b and d). Sampleswere incubated at 37° C. for 1 hour and immediately assayed for ureaseactivity (a and b), or dialyzed against 2% sucrose at 4° C. overnightand then assayed for activity c and d). The Mn⁺⁺ ion-activated ureaselost activity after dialysis. Activity was not restored by addition ofMn⁺⁺ ions, even after preincubation for several hours with Mn⁺⁺ ionbefore activity was assayed. The titration curves are normalized forzero time readings.

FIG. 13A is a graph showing that manganous ion has a synergistic effectand cupric, ferrous, and zinc ions have antagonistic effects on theactivation of recombinant H. pylori apourease by nickel ions andbicarbonate. Recombinant H. pylori apourease was incubated withactivation buffer containing 0.1 M HEPES buffer (pH 8.0), 0.3 M sodiumchloride, 1 mM EDTA, 100 μM Ni⁺⁺, and 200 mM sodium bicarbonate in theabsence or presence of other metal ions, as indicated in the Figure.After 2 hours of incubation at 37° C., 10 μl activated urease was addedto 1 ml urea broth, and activity was followed by monitoring increases inabsorbance at 550 nmn.

FIG. 13B is a graph showing the results of the same experiments as thosedescribed above for FIG. 13A, except that the activation buffercontained 500 μM Ni⁺⁺, rather than 100 μM Ni⁺⁺.

FIG. 14 is a photograph of a gel showing that use of a combination ofmanganous and Ni⁺⁺ ions results in a reduced amount of active urease, asdetermined by native PAGE and urease-specific silver staining.Recombinant urease activated under the conditions described above forFIG. 13A was stored at 4° C. overnight. The activity was monitored usingthe phenol red urea broth assay. The samples were mixed with an equalvolume of sample buffer, separated on a 4% PAGE gel under non-reducingand non-denaturing conditions, and activity staining was performed usingurease-specific staining conditions. Lane 1 contains jack bean urease,lane 2 contains sample incubated with Cu⁺⁺ and Ni⁺⁺ ions, lane 3contains sample incubated with Zn⁺⁺ and Ni⁺⁺ ions, lane 4 containssample incubated with Fe⁺⁺ and Ni⁺⁺ ions, lane 5 contains sampleincubated with Mg⁺⁺ and Ni⁺⁺ ions, lane 6 contains sample incubated withMn⁺⁺ and Ni⁺⁺ ions, and lane 7 contains sample incubated with Ni⁺⁺ ions.

FIG. 15 is a photograph of a gel showing that imidazole inhibits ureaseactivation by Ni⁺⁺ ions and bicarbonate. Recombinant H. pylori apoureasewas incubated at 37° C. for 1 hour and 53 minutes with activation buffercontaining 200 mM bicarbonate and 200 μM Ni⁺⁺. The samples were thenseparated by 6% native PAGE and stained for urease activity byurease-specific silver staining. Conditions used for the samplesfractionated in each of the lanes of the gel are as follows: lane 1, 25mM imidazole; lane 2, 12.5 mM imidazole; lane 3, 5 mM imidazole; lane 4,0.5 mM imidazole; lane 5, urease incubated with 5 mM MgCl₂, but no Ni⁺⁺.Lane 6 contains jack bean urease.

FIG. 16A is a graph showing that chemical modification of recombinant H.pylori apourease with iodoacetamide inhibits activation of urease withnickel ions and bicarbonate. Iodoacetamide was added to a recombinanturease solution (4 mg/ml) to a final concentration of 1 mM and incubatedat room temperature (Ure-IAA). At time zero, 100 μl of the Ure-IAA wasmixed with 100 μl of urease activation buffer and incubated at 37° C.(a). At five minutes, 250 μl of the (Ure-IAA) was transferred to aMacrosep 100 filter centrifuge, 5 ml of 2% sucrose was added, and thesample was concentrated to approximately 250 μl. The process ofdiafiltration was carried out two more times by adding 5 ml of 2%sucrose and concentrating the sample down to 250 μl. One hundred μl ofsample was mixed with 100 μl of urease activation buffer, and themixture was incubated at 37° C. for 1 hour (b). After 60 minutes ofincubation at room temperature, 100 μl of the Ure-IAA mixture was mixedwith 100 μl of urease activation buffer and incubated for 1 hour at 37°C. (c). At the 60 minute time point, 250 μl of the Ure-IAA mixture wasdiafiltered in 2% sucrose using the Macrosep 100, as described forsample b. One hundred μl of the diafiltered material was mixed with 100μl of urease activation buffer and incubated for 37° C. for 1 hour (d).Untreated urease (100 μl) was mixed with 100 μl of urease activationbuffer and incubated for 1 hour at 37° C. (e). At the end of 1 hour ofincubation of the urease in activation buffer, 10 μl was added to 1 mlof urea broth and the urease activity was monitored by following theincrease in absorption at 550 nm.

FIG. 16B is a graph showing the results of the same experiments as thosedescribed above for FIG. 16A, except that recombinant H. pylori ureasewas treated with DTNB (50 μl of DTNB (5 mg/ml) was mixed with 1 mlurease (4 mg/ml)). Samples a-e were treated exactly the same way asdescribed for the Ure-IAA mixture described above for FIG. 16A.

FIG. 17A is a graph showing the effect of N-ethyl maleimide on thesulfhydryl reactivity and in vitro activation of recombinant apourease.Recombinant apourease (3-4 mg/ml) in 20 mM Hepes buffer-2% sucrose (pH7.5) was incubated with 9 mM NEM for 60 minutes at room temperature.After incubation, excess reagent was removed by dialysis against 20 mMHEPES-2% sucrose at 2-8° C. The untreated urease was processedsimilarly. After dialysis, the DTNB-reactive sulfhydryl groups wereanalyzed. Urease samples were incubated with 0.3 mM DTNB solution andthe absorbance increase at 412 nm was monitored.

FIG. 17B is a graph showing measurements of enzymatic activity ofmodified and control urease (see description of FIG. 17A) incubated withbicarbonate/Ni⁺⁺ reagent using phenol broth, as described in above inreference to FIG. 1.

FIG. 18A is a set of graphs showing the effect of NEM treatment on themolecular stability of recombinant Helicobacter apourease, as studied byanalytical size-exclusion HPLC on a progel TSK-4000 column (5 mm×30 cmid) with GSXL guard column. Chromatography was carried out immediatelyafter modification and dialysis.

FIG. 18B is a set of graphs showing the effect of NEM treatment on themolecular stability of recombinant Helicobacter apourease, as studied byanalytical size-exclusion HPLC on a progel TSK-4000 column (5 mm×30 cmid) with a GSXL guard column. Chromatography of the modified, dialyzedsample was carried out after 15 days of storage at 2-8° C.

FIG. 19A is a graph showing that β-mercaptoethanol perturbs theUV-visible absorption spectrum of activated recombinant H. pyloriurease. Recombinant H. pylori urease was activated by incubation in 100mM HEPES, 1 mM EDTA, 0.3 M NaCl, 200 mM sodium bicarbonate, and 1 mMNi⁺⁺. After 1 hour of incubation at 37° C., the unbound Ni⁺⁺ andbicarbonate were removed by dialysis over two changes of 2% sucrose for24 hours at 4° C. The absorption spectrum is shown in the graph, and wasrecorded on a Shimadzu model UV2101 PC spectrophotometer. Spectrum (a)was recorded in the absence of β-mercaptoethanol, spectrum (b) wasrecorded in the presence of 1 mM β-mercaptoethanol, and spectrum (c) wasrecorded in the presence of 10 mM β-mercaptoethanol.

FIG. 19B is a graph showing the difference spectrum of activated ureasein the presence and absence of β-mercaptoethanol. Curve (1) is spectrum(b)-spectrum (a), and curve (2) is spectrum (c)-spectrum (a).

FIG. 20A is a series of graphs showing a comparison of the properties of(1) native H. pylori urease dialyzed in PBS, (2) native H. pylori ureasedialyzed in 50% glycerol, and (3) recombinant urease dialyzed in 50%glycerol, as analyzed by analytical size exclusion HPLC on a ProgelTSK-4000 column (5 mm×30 cm id) with a GSXL guard column. Recombinanturease from E. coli strain ORV214 and native H. pylori urease from theH. pylori strain CPM 630 were purified as is described further below.The purified urease was dialyzed against 10 mM phosphate, 0.15 M NaCl(pH 7.4), with or without 50% glycerol.

FIG. 20B is a photograph of native PAGE and Western blot analysis of thethree samples described above for FIG. 20A using antibody MPA3.

FIG. 20C is a photograph of isoelectric focusing analysis of the threesamples described above for FIG. 20A, carried out in a 5% polyacrylamidegel (pH 3-10).

FIG. 21A is a graph showing the isolation and characterization of fourdifferent forms of urease separated by analytical size exclusion HPLC.Recombinant urease solution was stored at 4° C. for 2-3 weeks. Thedifferent molecular forms of urease were separated by analytical sizeexclusion HPLC on a Progel-TSK 4000 SW×L column. The individual peakswere collected and analyzed. The different peaks are labeled as follows:P--high molecular weight polymeric urease (lower retention time inHPLC), O--octomeric urease, H--hexameric urease, and T--tetramericurease.

FIG. 21B is a photograph of reducing SDS-PAGE and Coomassie staininganalysis of the different molecular forms of urease separated byanalytical size exclusion HPLC, as described above for FIG. 21A.

FIG. 21C is a photograph of native PAGE and immunoblot analysis of thedifferent molecular forms of urease separated by analytical sizeexclusion HPLC, as described above for FIG. 21A using MPA3.

FIG. 21D is a UV-Visible absorption spectrum of the different molecularforms of urease separated by analytical size exclusion HPLC, asdescribed above for FIG. 21A.

FIG. 22A is a set of graphs showing the ELISA reactivity of differentforms of urease, isolated as described above for FIG. 21A, with MPA3 andMAB71.

FIG. 22B is a photograph of electron microscopic analysis of differentforms of recombinant urease. The different forms of urease were isolatedas described above for FIG. 21A. The different forms are labeled asfollows: P--polymeric urease, O--octomeric urease, H--hexameric urease,and T--tetrameric urease. Experimental details are described below inanalytical methods (page 51, lines 13-17).

FIG. 23A is a graph showing the effect of SDS on the SH group reactivityin a titration of sulfhydryl groups of recombinant urease. Recombinanturease (4.0 mg/ml) was titrated with DTNB (1 mM) in 100 mM Tris-HCl (pH8.0) in the absence or presence of 1% SDS. The absorbance at 412 nm isplotted against the time of the reaction.

FIG. 23B is a graph showing the effect of storage time on the SH groupreactivity in a titration of sulfhydryl groups of recombinant urease.Recombinant urease in 2% sucrose was stored at 4° C. for differentperiods of time. The DTNB titration was performed in the presence of 1%SDS, as described above.

FIG. 23C is a set of graphs showing the effect of DTNB treatment on themolecular state of urease, as analyzed by analytical size exclusionHPLC. Recombinant urease (4.0 mg/ml) in 100 mM Tris (pH 8.0) wasincubated with 1 mM DTNB for 1 hour at room temperature. TheDTNB-treated and control samples were then dialyzed against phosphatebuffered saline (pH 7.4) for 12-24 hours. Samples were subsequentlystored at 4° C. for 48 hours, and then analyzed by HPLC. The samplesused in the experiments shown in the three graphs are as follows: (a)recombinant urease solution in 2% sucrose, (b) DTNB-treated and dialyzed(PBS) urease, and (c) control urease dialyzed in PBS.

FIG. 24A is a photograph of reducing SDS-PAGE and Coomassie staininganalysis showing the effect of storage time and SH group blocking on theprotein profile of recombinant urease. DTNB-treated and control samplesdialyzed in PBS were prepared as described above for FIG. 23C.Recombinant urease stored for different time periods at 4° C. in 2%sucrose was also analyzed. The lanes of the gel are labeled as follows:lane MW, molecular weight markers; lane 1, urease solution stored at 4°C. for 9 weeks; lane 2, urease solution stored at 4° C. for 5 weeks;lane 3, urease solution stored at 4° C. for 3 10 days; lane 4, ureasesolution dialyzed against PBS for 48 hours; lane 5, urease solutiontreated with DTNB and then dialyzed against PBS for 48 hours.

FIG. 24B is a photograph of non-reducing SDS-PAGE and Coomassie staininganalysis showing the effect of storage time and SH group blocking on theprotein profile of recombinant urease. Sample preparation and lanelabels are as described above for FIG. 24A.

FIG. 24C is a photograph of native PAGE and Coomassie staining analysisshowing the effect of storage time and SH group blocking on the proteinprofile of recombinant urease. Sample preparation and lane labels are asdescribed above for FIG. 24A.

FIG. 25A is a photograph of Western blot analysis of urease stored underdifferent conditions and treated with DTNB using MPA3 (see analyticalmethods, below). The conditions used for the samples present in eachlane are as follows: 1, urease stored in solution for 9 weeks; 2, ureasesolution stored at 4° C. for 3 days; 3, urease solution dialyzed againstPBS; 4, DTNB-treated urease dialyzed against PBS.

FIG. 25B is a photograph of Western blot analysis of the urease samplesdescribed above for FIG. 25A using MPA4 (see analytical methods, below).

FIG. 25C is a photograph of Western blot analysis of the urease samplesdescribed above for FIG. 25A using MPA6 (see analytical methods, below)and non-reducing SDS-PAGE.

FIG. 26A is a graph showing that recombinant apourease obtained from anE. coli pellet carrying mutant ureA-ureB structural genes (H136Amutation) cannot be activated. Recombinant urease produced fromORV261-H136A mutant and ORV214 pellets were purified by a combination ofion-exchange and membrane filtration procedures, incubated withactivation buffer, and tested for activity, as described above for FIG.1.

FIG. 26B is a graph showing that recombinant apourease produced from E.coli strain ORV273, which carries mutant urease structural genes (K219Aplus H248A double mutation), is not activated by incubation withbicarbonate and nickel ions. Recombinant urease produced from the ORV273mutant and ORV214 pellets was purified by a combination of ion-exchangeand membrane filtration procedures, incubated with activation buffer,and tested for activity as described above in reference to FIG. 1.

FIG. 26C is a graph showing that recombinant apourease produced andpurified from mutant strain ORV273 is not activatable. Samples weretreated with in vitro activation and control buffers and then separatedon a native polyacrylamide gel. Gels were then stained for ureaseactivity using urease-specific silver staining. Lane 1 contains ureasefrom strain ORV214 incubated with in vitro activation buffer; lane 3contains urease from strain ORV214 incubated with bicarbonate in theabsence of nickel; lanes 5, 7, and 9 contain apourease from mutantstrain ORV273 incubated with activation buffer; and lane 11 containsnative H. pylori urease.

FIG. 26D is a schematic representation of the sites of mutation inORV273. Lys 219 and His 248 of native urease are both replaced with Alain ORV273.

FIG. 27 is a graph showing that recombinant apourease produced from theORV273 double mutant strain (rUre 96IO1) protects mice from H. pyloriinfection with an efficacy comparable to that for apourease produced bystrain ORV214 (rUre 94JO3). Groups of 10 mice were immunized with 4 oraldoses of either 50, 500, or 5000 ng of recombinant urease admixed with500 ng of E. coli heat-labile enterotoxin, and challenged with H.pylori. H. pylori colonization in the stomachs was determined byculture. Data points on the figure represent the number of colonyforming units of H. pylori recovered from a quarter of the gastricantral tissue of a mouse. The data show that immunization withrecombinant, mutant H. pylori urease (lot 96101 purified from ORV273)gave similar levels of protection as that produced with recombinant,wild type H. pylori urease (lot 94J03 purified from pORV214).

FIG. 28A is a graph showing that dinitrofluorobenzene (DNFB) treatmentblocks in vitro activation of recombinant apourease. Purifiedrecombinant urease from strain ORV214 (˜4.0 mg/ml) in 50 mM phosphate(pH 8.0) was incubated with 1 mM DNFB for 1 hour at 37° C. A controlsample, without DNFB, was also incubated for 1 hour at 37° C. After 1hour of incubation, 100 μl of DNFB-treated and control samples wereincubated with 100 μl of activation buffer containing Hepes, nickelchloride, and sodium bicarbonate for 2 hours and 30 minutes at 37° C.Urease enzyme activity was measured spectrophotometrically by monitoringthe formation of phenolate anion at 550 nm, as described in FIG. 1.Curve "a" is a control sample, curve "b" is an DNFB-treated sampleincubated with activation buffer, and curve "c" is a control sampleincubated with activation buffer in the presence of DNFB. These resultsshow that the DNFB-treated samples are not activated and that activationis blocked if DNFB is added along with activation buffer. There wasprecipitation in samples containing DNFB during incubation withactivation buffer.

FIG. 28B is a set of graphs showing that DNFB treatment stabilizesrecombinant apourease. Panel 1 shows DNFB treated urease and panel 2shows control, untreated urease. DNFB treated and control samples asdescribed above in reference to FIG. 28A were dialyzed against aphosphate buffer overnight. Dialyzed samples were analyzed by analyticalHPLC size-exclusion chromatography as described above. These resultsshow that molecular aggregation and degradation of control urease occursduring dialysis, and that DNFB treatment stabilizes the hexameric formof urease.

FIG. 28C is a graph showing that DNFB treatment for 5, 15, 30, and 60minutes blocks in vitro activation of recombinant apourease. Purifiedrecombinant urease from strain ORV214 was incubated with 0.5 mM DNFB.The reaction with DNFB was arrested at the indicated time points bytransferring 1 ml aliquots to 200 μl of 100 mM Cysteine-HCl (pH 7.0).Samples were then dialyzed overnight against 20 mM phosphate buffer, pH7.5, containing 2% sucrose. Dialyzed samples were then incubated withactivation buffer containing nickel chloride and bicarbonate for 2.5hours at 37° C. Urease catalytic activity was then monitored by usingthe phenol broth assay as described above in reference to FIG. 1.

FIG. 28D is a graph showing that the effect of DNFB on blocking in vitroactivation of urease is pH-dependent. Purified recombinant urease fromstrain ORV214 was incubated with 1 mM DNFB in 25-30 mM phosphate bufferat different pHs. After 5 minutes of incubation, samples were mixed withan equal volume of in vitro activation buffer containing nickel chlorideand sodium bicarbonate in HEPES buffer, and incubation was continued at37° C. for 3 hours. Urease enzyme activity was then determined asdescribed above in reference to FIG. 1.

FIG. 28E is a graph showing that DNFB inactivates pre-activated ureasein a pH-dependent manner, comparable to the effect of blocking in vitroactivation of urease. Purified recombinant apourease from strain ORV214was activated in vitro by incubation with bicarbonate-nickelchloride-Hepes buffer. Excess bicarbonate and nickel ions were removedby diafiltration against 10 mM phosphate, pH 7.4, using a Macrosep 100(100 kDa NMWCO) filter centrifuge. Activated urease was then mixed withan equal volume of 200 mM phosphate buffer at the indicated pHs andincubated with 1 mM DNFB for 1 hour. Urease activity was then determinedby using the phenol broth assay.

FIG. 29 is a graph showing that trinitrobenzene sulfonic acid (TNBSA)treatment blocks in vitro activation of recombinant apourease. Purifiedrecombinant apourease from strain ORV214 was incubated with 5 mM TNBSA,pH 8.2, for 30-120 minutes. Controls without TNBSA were incubated underidentical conditions. At specified time points, samples were taken,mixed with equal volumes of in vitro activation buffer containing nickelchloride, bicarbonate, and Hepes, and incubated at 37° C. for 2 hours.Urease activity was then determined by using the phenol broth assay, asdescribed above in reference to FIG. 1.

DETAILED DESCRIPTION

As is described below, Helicobacter apourease in solution can beheterogeneous (i.e., exist in different molecular forms), unstable,aggregated, interconverted between different molecular forms, andconverted from an enzymatically inactive form to an active form in vitroby incubation with bicarbonate and nickel ions.

Accordingly, the invention provides methods for stabilizing Helicobacterurease. In one example of these methods, urease is treated with acompound that modifies an amino acid of the urease so that it cannot beconverted into an enzymatically active form or the high molecularweight, polymeric aggregates characteristic of untreated urease (seebelow). An additional method for stabilizing urease, so that it cannotbe converted into an enzymatically active form, involves introducing agenetic modification (e.g., an amino acid substitution) into the urease.Urease stabilized using either of these methods can be used, forexample, in vaccination methods for preventing, treating, or diagnosingHelicobacter infection.

Chemical Stabilization of Helicobacter Urease

Each of the twenty amino acids has a free side chain, and many of theseside chains have reactive functional groups, such as the thiol group ofcysteine or the amino group of lysine. At least nine amino acid sidechains (Cys, Lys, Asp, Glu, Arg, His, Trp, Tyr, and Met) can react undermild conditions with quite specific reagents to yield chemicallymodified amino acid derivatives. Numerous specific reagents formodifying amino acid side chains are known in the art (see, e.g.,Fagain, Biochimica et Biophysica Acta 1252:1-14, 1995; Means et al.,Bioconjugate Chem. 1:2-12, 1990; Imoto etal., in Protein Function: aPractical Approach (Creighton, ed.) 247-277, IRL Press, Oxford, 1989;Lundblad et al., Chemical Reagentsfor Protein Modification, Vol. 1 & 2,CRC Press, Boca Raton, Fla., 1984), and can be used in the invention tostabilize Helicobacter urease.

Urease polypeptides that can be stabilized using the methods of theinvention include urease polypeptides that are purified fromHelicobacter (e.g., H. pylori or H. felis) cultures (Michetti et al., WO94/09823; Dunn et al., J. Biol. Chem. 265:9464-9469; also see below), aswell as urease polypeptides that are produced using recombinant methods(e.g., recombinant apourease; Lee et al., J. Infect. Dis. 172:161-172,1995; Hu et aL, Infect. Immun. 60:2657-2666, 1992; also see below).Though there may be no differences in the amino acid sequences of anative urease and a corresponding recombinant apourease lacking nickelions, the lack of nickel ions at the active site of the apourease mayaffect the conformation of the protein, particularly in the active siteand nearby regions. Thus, the accessability and reactivity of functionalamino acid residues in the active site of the apoprotein is likely to bevery different from those of the native protein. The experimentsdescribed below show that chemical reagents used for amino acidmodification can react with recombinant apourease and influence theactivation and stability of the apourease.

Examples of chemical compounds that can be used to stabilize urease areas follows. Compounds that modify thiol groups, includingdisulfide-reactive agents, such as 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB), 2,2'-dithiodipyridine (DTDP), cystine, cystamine, methylmethanethiolsulfonate (MMTS), and dinitrofluorobenzene (DNFB), as wellas alkylating agents, such as iodoacetate (IAA), iodoacetamide (IAM;this compound also modifies lysine and histidine residues), andN-ethylmaleimide (NEM), can be used. Additional compounds that modifythiol groups and can be used in the invention include proton pumpinhibitors that are converted into thiol-reactive sulfenamides at lowpH. Examples of such compounds include omeprazole, lansoprazole, andderivatives of these compounds (e.g., AG-2000; Nagata et al.,Antimicrobial Agents and Chemotherapy 39(2):567-570, 1995).

Compounds that modify amino groups can also be used to stabilize urease.For example, acetylating agents, such as acetic anhydride, methyl acetylphosphate, pyridoxal phosphate, fluoronitrobenzenes (e.g.,dinitrofluorobenzene (DNFB)), and LAM can be used. Trinitrobenzenesulfonic acid (TNBSA), which also modifies amino groups, can also beused to stabilize urease. In addition, compounds that modify histidine(e.g., iodoacetic acid and iodoacetamide) or arginine (phenylglyoxal andglyoxal) residues can be used in the invention to stabilize urease.

Methods for using these and other compounds to modify amino acids areknown in the art and can readily be adapted for use with urease by oneskilled in the art. For example, in the case of IAA, urease (e.g., 4mg/ml) can be incubated with 0.1-25 mM IAA, e.g., 1 mM IAA, at roomtemperature for 5-20 minutes. Amounts of other compounds that can beused to stabilize urease are as follows: 1-200 mM LAM, 5-200 mM NEM,0.1-5.0 mM DTNB, 0.01-4.5 mM DTDP, 0.5-1.0 mM Cystine, 0.25-1.0 mMCystamine, and 25-100 μM MMTS. Stabilization reactions can be carriedout, for example, in 80 mM HEPES (pH 7.75), 8 mM EDTA, at 20-40° C.,e.g., at room temperature or 37° C.

Appropriate reaction conditions (e.g., reaction volume, buffer,incubation temperature, incubation length) for use with any of theabove-listed compounds can readily be determined by one skilled in theart. For example, 0.5-10 mg/ml (e.g., 3-4 mg/ml) urease can be incubatedwith 0.1-100 mM modifying agent. Additional examples of reactionparameters that can be used are set forth below.

Chemical modification of urease can be performed after the final step ofpurification. For example, purified urease in a Tris or phosphate buffer(pH 7.5-8.6) can be incubated with 1-5 mM Iodoacetamide, 1-10 mM NEM, or0.3-1 mM DTNB for 1 hour at room temperature. After this incubation, thesamples can be diafiltered into 2% sucrose, with or without buffer, andconcentrated to about 4.0 mg/ml.

Genetic Stabilization of Helicobacter Urease

In addition to the chemical methods described above, urease polypeptidescan be stabilized, in order to prevent activation (e.g., in vitroactivation), using genetic modification methods. Methods for modifying anucleic acid so that it encodes an amino acid sequence containing amodification (i.e., an amino acid substitution, deletion, or addition),for example, site-directed and PCR mutagenesis methods, are well knownin the art (see, e.g., Ausubel et al., eds. Current Protocols inMolecular Biology, Wiley & Sons, New York, 1989). A nucleic acidmodified using these methods can be used to produce the modified proteinusing standard expression methods, such as those described below (alsosee, e.g., Ausubel et al., supra).

Amino acids that can be modified in order to produce stabilizedHelicobacter urease of the invention include, for example, residues thatare at the active site of the molecule (e.g., lysine 219), as well asresidues that are involved in the formation of disulfide bridges.Specific examples of modifications included in the invention are asfollows. A histidine residue at position 136, 138, 221, 248, 274, 314,322, or 323 can be replaced, for example, with alanine or leucine. Theaspartate residue at 362 or the lysine residue at position 219 can bereplaced with alanine or leucine. In addition, cysteine (e.g., Cys 321and Cys 257) and arginine (e.g., Arg 338 and Arg 340) residues of ureasecan be modified (for example, substituted, e.g., with alanine orleucine) according to the invention.

Use of stabilized Urease in Methods for Preventing or TreatingHelicobacter Infection

Urease that has been stabilized using the methods described above can beformulated for administration using standard methods appropriate for theintended mode of administration. For example, the stabilized urease canbe combined with a stabilizer (e.g., a carbohydrate mannitol) and theproduct freeze dried (i.e., lyophilized). This process further preventsdegradation by aggregation and fragmentation. In addition, the productis stable for months following lyophilization.

Stabilized urease can be freeze-dried following the final purificationstep (see below). The purified protein product (approximately 4 mg/ml)is dialyzed against 2% sucrose or diafiltered using a 10-100 kDa NMCOdiafiltration membrane, and this solution is transferred tolyophilization vials. The vialed solution is either frozen in liquidnitrogen and then placed into the lyophilizer, or cooled to 4° C. andthen placed in the lyophilizer, where it is frozen to -40° C., or lower.Lyophilization is carried out using standard methods. The freeze-driedproduct can be reconstituted in water.

Stabilized urease can be administered to a mucosal surface of a patient,such as a human patient, in order to stimulate a mucosal immune responseeffective to provide protection to subsequent exposure to Helicobacterand/or facilitate clearance of a pre-existing Helicobacter infection.Preferably, stabilized urease is administered to elicit a mucosal immuneresponse associated with production of anti-urease IgA antibodies and/orinfiltration of lymphocytes into the gastric mucosa. The stabilizedurease can be administered to any mucosal surface of the patient.Preferable mucosal surfaces are oral, intranasal, and rectal (e.g., byuse of an anal suppository) surfaces. In addition to being administeredto a single mucosal surface, the vaccine of the invention can beadministered to combinations of mucosal surfaces (e.g., oral+rectal,oral+intranasal, or rectal+intranasal) or a combination of mucosal andparenteral administration can be used. In the case of oraladministration, it is preferable that the administration involvesingestion of the vaccine, but the vaccine can also be administered as amouth wash, so that an immune response is stimulated in the mucosalsurface of the oral cavity, without actual ingestion of the vaccine.Alternatively, stabilized urease can be administered to a patient by theparenteral route, e.g., by subcutaneous, intravenous, or intramuscularinjection.

Appropriate dosages of stabilized urease administered to a patient,whether for prevention or treatment of Helicobacter infection, can bedetermined by one skilled in the art. Generally, dosages will containbetween about 10 μg to 1,000 mg stabilized urease. For mucosalimmunization, preferred doses are between about 10 mg and 100 mg, whilefor parenteral immunization, preferred doses are between about 10 μg and100 μg.

At least one dose of the stabilized urease can be administered to thepatient, for example, at least two, four, six, or more total doses canbe administered. It may be desirable to administer booster doses of thestabilized urease at one or two week intervals after the lastimmunization. Generally one booster dose containing less than, or thesame amount of, stabilized urease as the initial dose is administered.For example, the vaccine regimen can be administered in four doses atone week intervals. For mucosal immunization, priming and booster dosescan be administered to the same or different mucosal surfaces. In thecase of different mucosal surfaces, for example, an oral priming dosecan be followed by intranasal or rectal boosters, an intranasal primingdose can be followed by oral or rectal boosters, or a rectal primingdose can be followed by oral or intranasal boosters.

Stabilized urease can be co-administered with an adjuvant. For mucosalimmunization, any mucosal adjuvant known in the art that is appropriatefor use in the patient can be used. For example, the mucosal adjuvantcan be cholera toxin (CT), enterotoxigenic E. coli heat-labile toxin(LT), or a derivative, subunit, or fragment of CT or LT that retainsadjuvant activity. The mucosal adjuvant is co-administered withstabilized urease in an amount effective to elicit or enhance an immuneresponse, particularly a humoral and/or a mucosal immune response. Theratio of adjuvant to stabilized urease that is administered can bedetermined by standard methods by one skilled in the art. For example,the adjuvant can be present at a ratio of 1 part adjuvant to 10 partsstabilized urease. Stabilized urease can be co-administered by theparenteral route with one or more of many adjuvants or immunomodulatorsknown in the art. For example, the parenteral adjuvant can be aluminumhydroxide, aluminum phosphate, calcium phosphate, muramyl tripeptide,muramyl dipeptide, immunostimulatory complexes (ISCOMs), saponinderivatives, such as QS 21, oil-in-water emulsions, liposomes, blockpolymers, or any combinations of the above.

A buffer can be administered prior to administration of stabilizedurease, in order to neutralize or increase the pH of the gastric acid ofthe stomach. Any buffer that is effective in raising the pH of gastricacid and is appropriate for use in the patient can be used. For example,buffers, such as sodium bicarbonate, potassium bicarbonate, and sodiumphosphate, can be used. In the case of oral administration, the vaccinecan be buffer-free, meaning that a pH-raising buffer compound effectiveto significantly affect gastric acid pH is not administered to thepatient either prior to, or concomitant with, administration of thevaccine.

The vaccine formulation containing stabilized urease can also containany of a variety of other components, including stabilizers, flavorenhancers (e.g., sugar), or, where the vaccine is administered as anantibacterial therapeutic, other compounds effective in facilitatingclearance and/or eradication of the infecting bacteria (e.g., antibioticcompounds and proton pump inhibitors).

For prophylactic therapy, the vaccine containing stabilized urease canbe administered at any time prior to contact with, or establishment of,Helicobacter infection. Because the vaccine can also act as anantibacterial therapy, there is no contraindication for administrationof the vaccine if there is marginal evidence or suspicion of apre-existing Helicobacter infection (e.g., an asymptomatic infection).

For use of the vaccine in antibacterial therapy, stabilized urease canbe administered at any time before, during, or after the onset ofsymptoms associated with Helicobacter infection or with gastritis,peptic ulcers or other gastrointestinal disorders. Although it is not aprerequisite to the initiation of therapy, one can confirm diagnosis ofHelicobacter infection by, e.g., a ¹³ C breath test, serology,gastroscopy, biopsy, or another Helicobacter detection method known inthe art. The progress of immunized patients can be followed by generalmedical evaluation, screening for Helicobacter infection by serology, ¹³C. breath test, and/or gastroscopic examination.

In addition to its use for immunization against Helicobacter, stabilizedurease can be used to immunize animals or humans against otherconditions, such as ulcerative colitis and ammonia toxicity associatedwith hepatic failure.

Experimental Results

In Vitro Activation of Recombinant Helicobacter pylori ApoureaseExpressed in Escherichia coli by Bicarbonate and Nickel Ions

Recombinant H. pylori urease apoprotein expressed in E. coli strainORV214 can be used as an oral vaccine for the prophylactic andtherapeutic treatment of peptic ulcer caused by H. pylori infection.Since it does not contain Ni⁺⁺ ions and is synthesized by recombinant E.coli lacking urease accessory assembly genes, the urease apoprotein ispredicted to be catalytically inactive. The experiments described belowshow that recombinant H. pylori urease apoprotein expressed in, andpurified from, the ORV214 E. coli strain can be activated by in vitroincubation with supra-physiological concentrations of Ni⁺⁺ andbicarbonate.

Activation of recombinant urease with Ni⁺⁺ and bicarbonate

Urease enzyme causes cleavage of urea as follows:

    H.sub.2 N--CO--NH.sub.2 +H.sub.2 O→2NH.sub.3 +CO.sub.2

Urease activity was detected in urease preparations that werepre-incubated with bicarbonate and Ni⁺⁺ ions using the assay methodsdescribed below. The activation effect of bicarbonate and Ni⁺⁺ ions onrecombinant urease measured using the phenol red urea broth colorimetricmethod is shown in FIG. 1. The conversion of urea to ammonia and carbondioxide results in an increase in pH of the medium. The assay mixturecontains phenol red, which undergoes ionization in alkaline pH to form apink colored product, as measured by optical density readings at 550 nm.The reactions are performed in 1 cm cuvettes and increases in absorbanceat 550 nm are continuously monitored using a Shimadzu Model UV-2101 PCwith a CPM 260 multi-channel, temperature-controlled cuvette holder.Activity was detected only when both bicarbonate and Ni⁺⁺ ions werepresent in the pre-incubation system. These results are consistent withobservations of K. aerogenes urease activity (Park et al., Science267:1156-1158, 1995). Formation of ammonia was confirmed by directestimation of ammonia from urea. Detection of urease activity by nativePAGE and urease-specific silver staining is shown in FIG. 2. The ureaseprotein is fractionated by electrophoresis in a polyacrylamide gel undernon-denaturing conditions. The gel is then incubated with urea andstained with a pH sensitive redox system consisting of hydroquinone andp-aminophenol, followed by incubation with silver nitrate. Metallicsilver is deposited on the gel at sites of urease enzymatic activity.

Time dependence of activation

A time course of urease activation is shown in FIG. 3. These resultsshow that activation is a time-dependent process, consistent with theoccurrence of time-dependent modification of the protein.

Ni⁺⁺, bicarbonate, temperature, and pH-dependence of activation

Experiments showing the Ni⁺⁺ ion concentration dependence of ureaseactivation are shown in FIG. 4. Activation increases with increasingNi⁺⁺ ion concentrations. Under the experimental conditions used, Ni⁺⁺ion concentrations at or below 20 μM in the pre-incubation system didnot cause any significant activation of recombinant urease. In separateexperiments, activation of recombinant urease was observed to occur atNi⁺⁺ concentrations of 50 μM and 100 μM.

Experiments showing the dependence of activation on bicarbonateconcentration are shown in FIG. 5. The extent of activation increasedwith increasing bicarbonate concentrations, within the range ofconcentrations tested (5-500 mM bicarbonate).

Experiments showing the urease activity-specific silver staining ofrecombinant urease activated with different concentrations of Ni⁺⁺ andbicarbonate are shown in FIG. 6, and experiments showing the effect oftemperature on the activation of recombinant urease are shown in FIG. 7.Activation is a temperature-dependent process, with the extent ofactivation increasing with increasing temperatures, ranging from 4-40°C. The slope of the best linear portion of the time course kinetics wasassumed to be a relative measure of the activity. Because the substrateconcentration in the assay mixture was far above the saturatingconcentration, the activity measured was very close to the maximalvelocity, and hence is a measure of the apparent turnover rate constant.An Arrhenius plot of the activity resulted in a straight line, and anenergy of activation of 17.4 kcals was calculated for urease activation.

pH dependence of activation

The results of an experiment showing the pH dependence of ureaseactivation is shown in FIG. 8. No detectable activation occurred at pH6.5. In separate experiments, it was found that activation does notoccur at lower pH (<6.0).

Urease binding affinity of activated recombinant urease

The activated urease retained catalytic activity after dialysis ofunbound Ni⁺⁺ ions and bicarbonate. The activity of the dialyzed samplewas retained for at least 1 week after storage at 4° C. in 2% sucrose(FIG. 9). The binding affinity of activated urease for urea wasdetermined by carrying out spectrophotometric measurements of thedecrease in NADH absorption at 340 nm accompanying NADH-dependentglutamate dehydrogenase catalyzed conversion of ammonia, produced by thehydrolysis of urea, and α-ketoglutarate to L-glutamic acid. Theglutamate dehydrogenase reaction utilizes NADH that is converted to NAD.The NADH to NAD conversion is accompanied by a decrease in absorption at340 nm. The reactions are performed in 1 cm cuvettes and decreases inabsorbance at 340 nm are continuously monitored using a Shimadzu ModelUV-2101 PC with a CPM 260 multi-channel, temperature-controlled cuvetteholder.

Initial velocities computed from the slopes of the linear portions ofthe curves showing the kinetics of decreases in 340 nm absorption atvarying concentrations of urea were plotted in Lineweaver-Burk doublereciprocal plots. The Lineweaver-Burk plot for reactivated recombinantH. pylori urease is compared with that for jack bean (type IV (EC3.5.1.5) Sigma catalog # U2000, Lot 122H7115) urease in FIG. 10. TheK_(m) value of 1.1 mM calculated for the recombinant urease is slightlyhigher than the values reported for native H. pylori urease (Mobley etal., Microbiol. Rev. 59:451-480, 1995), but is within the limits ofexperimental variations. The value is nearly four-fold lower than thatestimated for jack bean urease under similar conditions.

Effect of other metal ions and imidazole on activation of recombinanturease

Ni⁺⁺, Mn⁺⁺, Zn⁺⁺, Cu⁺⁺, Fe⁺⁺, and Mg⁺⁺ ions were tested for theirabilities to activate recombinant urease. Pre-incubation of recombinanturease with Ni⁺⁺ ions or Mn⁺⁺ ions resulted in activation of urease, asmonitored by its ability to cause an increase in pH when incubated withurea in a phenol red urea broth assay (FIG. 1). Activation ofrecombinant urease by Mn⁺⁺ ions was also demonstrated by directestimation of liberated ammonia using Nessler's reagent. However, Mn⁺⁺ion-activated recombinant urease did not result in detection of a bandhaving urease activity, as detected by native PAGE and byurease-specific silver staining. Also, while recombinant ureaseactivated with Ni⁺⁺ ions and bicarbonate retained catalytic activityafter dialysis of unbound Ni⁺⁺ ions and bicarbonate, the Mn⁺⁺ion-activated recombinant urease lost catalytic activity after dialysis(FIG. 12). In a separate experiment, it was observed that Li⁺⁺ and Co⁺⁺ions do not cause urease activation.

Combinations of Ni⁺⁺ and other metal ions were tested to see whether theother metal ions can interfere with the activation of recombinant ureaseby Ni⁺⁺ (FIG. 13). Ni⁺⁺ ions (0.1 mM) and 1 mM of the other metal ionswere used. Under these conditions, Mg⁺⁺ ions had no detectable effect.Mn⁺⁺ ions enhanced the activation of recombinant urease, showing thatthere is a synergistic effect of Ni⁺⁺ and Mn⁺⁺ ions. Cu⁺⁺, Fe⁺⁺, andZn⁺⁺ ions inhibited activation of recombinant urease by Ni⁺⁺. In aseparate experiment, it was found that Co⁺⁺ ions also inhibit activationwith Ni⁺⁺ and bicarbonate. The synergistic effect of Ni⁺⁺ was notdetected when the pre-incubation system contained a higher concentrationof Ni⁺⁺ (500 EM). Native PAGE and urease-specific silver staining ofurease activated with Ni⁺⁺ and the combinations of other metal ionsconformed the inhibitory effect of Cu⁺⁺, Fe⁺⁺, and Zn⁺⁺ ions on ureaseactivation. However, the activation effect of Mn⁺⁺ was not detected byanalysis of the stained gel. In contrast, the activity in the Mn⁺⁺ andNi⁺⁺ ion-activated system was less than that of Ni⁺⁺ alone or Ni⁺⁺ andMg⁺⁺ (FIG. 14), indicating that the fraction of urease that had Mn⁺⁺ions in the active site had lost the activity during electrophoresis.This observation is consistent with the results obtained with Mn⁺⁺ ions.The active site of urease is known to contain a cluster of histidineresidues that serve as the binding site for Ni⁺⁺. Imidazole is known tocomplex with Ni⁺⁺, and hence should inhibit the binding of Ni⁺⁺ to theactive site of recombinant urease. Imidazole concentrations ranging from2.5-25 mM were found to inhibit the activation of recombinant urease ina concentration-dependent manner (FIG. 15).

Effect of iodoacetamide and DTNB on activation of recombinant urease

Chemical modification of recombinant urease using iodoacetamide or5-5'-dithio-bis-2-nitrobenzoic acid (DTNB) has a stabilizing effect onurease in solution, showing that sulfhydryl groups play a role inmaintaining the molecular state of recombinant urease in solution. Itwas observed that recombinant urease alkylated by iodoacetamide or DTNBis not activated with Ni⁺⁺ and bicarbonate, and that includingiodoacetamide or DTNB in the activation mixture also resulted in theblocking of activation. In addition, iodoacetamide or DTNB-modifiedurease, after removal of excess modifying agent, was also not activated(FIGS. 16A and 16B).

N-ethyl maleimide is another sulfhydryl group modification reagent.Incubation of recombinant apourease (3-4.0 mg/ml) in 20 mM Hepes buffer(pH 7.5) with 9 mM NEM at room temperature for 60 minutes resulted inblocking of the sulfhydryl groups, as determined by reactivity with thethiol-specific reagent DTNB (FIG. 17A). The modified protein was notactivated by bicarbonate and nickel, while the unmodified, controlurease was activated under identical conditions (FIG. 17B). Afterfifteen days of storage at 2-8° C., the control (unmodified apourease)was aggregated and degraded significantly, as shown by a loss of morethan 50% of urease by HPLC analysis (the loss being interpreted as dueto removal of aggregates during filtration of the sample through a 0.2μm filter before HPLC), while the NEM modified urease retained itsmolecular integrity (FIGS. 18A and 18B).

Park et al. (Science 267:1156-1158, 1995) have proposed thatcarbamylation of lysine 217 in K. aerogenes apourease, which is in asimilar location as lysine 219 in Helicobacter urease, could be involvedin the mechanism of in vitro activation of urease. We have used severalamino group modifying reagents to chemically modify recombinantHelicobacter apourease and we have tested the effect of suchmodifications on in vitro activation. For example, modification ofapourease with dinitrofluorobenzene (DNFB) blocked in vitro activation(FIGS. 28A-28C).

The effect of DNFB was pH dependent, as the modification was moreeffective at alkaline pH (pH>7.5) than at neutral or acidic pH (FIG.28D). This is consistent with the proposed reactivity of lysine 219.Similar inactivation of enzymatic activity was noted for an alreadyactivated urease (FIG. 28E). This result shows that modification oflysine 219 may not be the cause of in vitro activation by DNFBmodification. DNFB can also react with cysteine residues and otherfunctional residues, and this could explain the effect of DNFB onapourease and reactivated urease.

Trinitrobenzene sulfonic acid (TNBSA, 5 mM) effectively blockedapourease from being reactivated in vitro (FIG. 29). Under similarconditions, incubation of in vitro activated urease with 5 mM TNBSA didnot inactivate the enzymatic activity. This observation shows that alysyl group modification in apourease may result in blocking of in vitroactivation and the group in apourease modified by TNBSA is not availablein activated urease. This observation is consistent with thecarbamylation of a lysine residue being involved in the mechanism for invitro activation.

In order to obtain more evidence for the binding of Ni⁺⁺ to recombinanturease, activated urease was prepared by incubation of recombinanturease with activation buffer containing Ni⁺⁺ and bicarbonate. Excessbicarbonate and Ni⁺⁺ ions were removed by extensive dialysis against 2%sucrose. The activated urease was then concentrated using a Macrosep 100filter centrifuge (Filtron, Inc.) to a concentration of 10 mg/ml, asdetermined by spectrophotometric analysis at OD₂₈₀. The absorbancespectrum of the urease was recorded in the presence and absence ofβ-mercaptoethanol, and in the presence of β-mercaptoethanol, twodistinct absorption maxima were detected at 410-407 mn and 330-325 nm(FIGS. 19A and 19B).

Recombinant H. pylori urease was purified from E. coli carrying clonedurease structural subunit genes ureA and ureB. The recombinant proteinwas structurally and immunologically identical to native H. pyloriurease, but had no detectable enzymatic activity. Four molecular formsof urease, designated tetrameric, hexameric, octomeric, and higherpolymeric forms, were isolated from urease stored in solution. Theoctomeric and hexameric forms also were enzymatically inactive andshowed similar immunoreactivity with a mouse anti-H. pylori ureasepolyclonal antibody, MPA3, and an anti-H. felis urease B monoclonal IgA,MAB71. Electron microscopic examination revealed that the differentmolecular forms appear as particles of different sizes. There was adecrease in the total number of free sulfhydryl groups in recombinanturease solutions stored at 4° C., as compared to freshly preparedurease. Blocking free sulfhydryl groups of recombinant urease with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) or iodoacetamide was shown toreduce the molecular associations. Consistent with these observations,β-mercaptoethanol stabilized urease in the hexameric form. These resultsdemonstrate the existence of inter-subunit disulfide linkages betweendifferent UreB subunits, UreA subunits, and UreA and UreB subunits.These results are described further, as follows.

Comparison of the properties of native and recombinant H. pylori urease

Recombinant H. pylori urease-producing E. coli strain ORV214, containingstructural genes encoding UreA and UreB, was constructed in an IPTGinducible expression system (Lee et al., J. Infect. Dis. 172:161-172,1995; Pappo et al., Infect. Immun. 63:1246-1252, 1995). The bacteriawere grown in 40 or 400 liter fermentation tanks in production mediacontaining yeast extract, tryptone, and 0.6-1.5% glycerol withoutantibiotics. After 16-24 hours of induction, the bacteria wereharvested, washed by centrifugation, and stored at -20° C. Recombinanturease was purified from the frozen bacteria by a procedure involvingbacterial lysis using a microfluidizer, clarification by centrifugation,and a purification procedure involving the use of non-adsorptiveDEAE-Sepharose, ultrafiltration-diafiltration (100 kDa MW cut-off, Omegamembranes, Filtron, Inc.), adsorptive DEAE-Sepharose chromatography,ultrafiltration/diafiltration, and non-adsorptive Q-Sepharose anionexchange chromatography. The urease that did not bind to the Q-Sepharoseunder the chromatographic conditions was buffer-exchanged into 2%sucrose.

The urease purified using the above-described protocol was visualized as29 kDa (UreA) and 60 kDa (UreB) protein bands by Coomassie staining of areducing SDS-PAGE gel. Densitometric scanning showed that the proteinwas more than 95% pure. Both the UreA and UreB subunits reacted inWestern blot analysis with the mouse polyclonal antibody MPA3, which wasraised against purified H. pylori urease. The mouse polyclonal antibodyMPA6, which was raised against the UreB subunit of H. pylori urease,reacted with the UreB subunit, and the mouse polyclonal antibody MPA4,raised against the UreA subunit of H. pylori urease, reacted with theUreA subunit of the recombinant urease. N-terminal amino acid sequencingof the reducing SDS-PAGE-separated UreA and UreB subunits of therecombinant urease was compared with the theoretical sequence derived bytranslation of the sequences of the genes encoding UreA and UreB(Clayton et al., Infect. Immun. 57:623-629, 1989). There was 100% aminoacid sequence identity, for the 25 residues analyzed, between the actualsequence and the predicted sequence. These results established theidentity of the purified recombinant urease. The amino acid compositionof recombinant urease is compared with those reported for otherbacterial and plant ureases in Table 1.

Analytical size exclusion HPLC revealed that urease purified accordingto the above-described protocol consisted of a major peak (80-90% of thetotal area) with a peak retention time of 9.1-9.4 minutes under thechromatographic conditions used (FIG. 20A, chromatogram 3). The HPLCcolumn was calibrated using calibration markers from Pharmacia-LKB. Theapparent molecular weight of this peak is between 550-600 kDa. This peakwas designated as the hexameric form of urease (UreA-UreB)₆. Anotherminor peak, with a retention time of 8.3-8.5 minutes, was also detected.This peak contributed 5-20% of the total area in different preparations.This form, having a molecular weight of 700 kDa, was designated as thehigher molecular weight, octomeric form of urease. Both of the formswere isolated from the analytical size-exclusion HPLC and identified tobe different forms of urease by SDS-PAGE, native PAGE, Coomassiestaining, Western blot analysis, and ELISA using polyclonal andmonoclonal anti-urease antibodies (FIG. 21).

The properties of purified recombinant urease were compared with thoseof H. pylori urease purified from the bacterial strain CPM 630. Thenative urease has a slightly lower molecular weight, as detected byanalytical size exclusion HPLC (FIG. 20A, chromatogram 1). A minor, highmolecular weight protein peak was detected in native urease stored inPBS (FIG. 20A, chromatogram 2). This peak was absent in freshly purifiedurease or urease dialyzed against PBS containing 50% glycerol and storedat -20° C. This high molecular weight component was analogous to theminor, high molecular weight urease peak seen with recombinant urease.In non-reducing SDS-PAGE, the recombinant urease, as well as the nativeurease stored in PBS at 4° C., showed a small amount of high molecularweight protein bands that reacted with anti-ureases antibodies. Thenative urease and recombinant urease were shown to have identicalmorphologies by electron microscopic examination (Lee et al., J. Infect.Dis. 172:161-172, 1995). Native and recombinant urease showed comparableELISA reactivity with MPA3 and MAB71. The native PAGE and Coomassiestaining, as well as the Western blot analysis with MPA3 (FIG. 20B), ofnative urease showed discrete high molecular weight protein bands (lanes1 and 2), but recombinant urease (lane 3) showed more heterogeneity. Inisoelectric focusing (IEF) gels, native urease migrated as a sharpprotein band (FIG. 20C, lanes 1 and 2) with a pI value of 6.1-6.15. Therecombinant urease had a pI value of 6.25-6.35 and appeared to be moreheterogenous (FIG. 20C, lane 3). These results show that native ureaseand recombinant apourease lacking nickel have apparently similarphysicochemical and immunochemical properties. The recombinant ureaseapoenzyme did not exhibit any urease enzymatic activity, and suchactivity was not inducible by in vitro incubation of the protein withnickel ions. Activity was induced by incubation of recombinant ureasewith nickel and bicarbonate.

Stability of recombinant urease under different conditions

The purified, recombinant H. pylori urease was stable for at least oneyear when freeze-dried and stored at -20° C., as evaluated by analyticalsize exclusion HPLC. The protein retained its molecular integrity, asdetermined by reducing SDS-PAGE, Coomassie staining, and densitometry,as well as by Western blot analysis using MPA3. The protein alsoretained immunoreactivity, as detected by ELISA analysis, using MPA3 andMAB71.

Molecular changes occurred in recombinant urease solutions stored at 4°C. under some conditions. Molecular association occurred initially.Subsequently, the solutions became turbid and the protein precipitated.After initial molecular association and precipitation, breakdownproducts were also detected. In 2% sucrose at 4° C., the area of thehexameric urease peak (H) decreased with time, with an increase in thearea of the high molecular weight, octomeric urease (0) peak at 8.3minutes. After a few days, the molecule started to breakdown to a lowmolecular weight peak, with a retention time of 10.1-10.3 minutes,corresponding to a putative pentameric or tetrameric (T) urease. A highmolecular weight, soluble, polymeric form (P) that eluted in the voidvolume of the column (retention time of approximately 6 minutes) wasalso frequently detected. A chromatographic profile of a recombinanturease solution stored at 4° C. for one month is shown in FIG. 21A.Reducing SDS-PAGE and Coomassie staining showed no difference in profilebetween freshly prepared and stored urease solutions. Only UreA and UreBsubunits were detected. However, native gel analysis showed thatmolecular changes took place in the course of storage. Non-reducingSDS-PAGE revealed a number of high molecular weight bands in addition toUreA and UreB subunits. In a freshly prepared sample, more than 80% ofthe protein was in the UreA and UreB forms, suggesting that there werelittle inter-subunit covalent disulfide bonds. However, in the samplesstored in solution for different, longer time periods, the intensity ofUreA and UreB bands decreased significantly, while the intensity of highmolecular weight components increased. Those high molecular weightcomponents reacted with anti-UreB, as well as anti-UreA, antibodies inWestern blot analysis. These results showed that the high molecularweight components contain both UreA and UreB subunits held together bycovalent inter-subunit disulfide bonds. The urease could also bestabilized by storage in 50% glycerol at -20° C.

Biochemical and immunochemical properties of the different forms ofurease

Because different molecular forms of urease were detected bysize-exclusion HPLC analysis of urease stored in solution, these formswere separated and the biochemical and immunochemical characteristics ofthe forms were studied by reducing SDS-PAGE (FIG. 21B), native PAGE,Western blot analysis (FIG. 21C), UV-visible absorption spectroscopy(FIG. 21D), ELISA using different anti-urease antibodies (FIG. 22A), andelectron microscopy (FIG. 22B). The reducing SDS-PAGE and Coomassiestaining showed that the different peaks all contain UreA and UreBsubunits. Native PAGE and immunoblot analysis revealed the presence ofmultiple urease bands with different molecular weights (FIG. 21C). Thepeaks with lower retention times in HPLC showed higher proportions ofhigh molecular weight protein bands (FIG. 21C, lane P), and peaks withhigher retention times showed low molecular weight protein bands innative PAGE (FIG. 21C, lane T). These results showed that the differentpeaks of urease separated by HPLC represent different molecular forms ofurease. The absorption spectrum of the different peaks of ureaseseparated by HPLC is shown in FIG. 21D. While the spectral properties ofthe peaks with retention times of 8.3 (octomer), 9.3 (hexamer), and 10.1(tetramer) minutes are apparently similar, the spectral properties ofthe higher molecular weight form that eluted in the void volume of thecolumn was strikingly different. The absorption spectrum of thepolymeric form suggests significant contribution by Raleigh lightscattering. The observed spectrum of the polymeric (P) form is analogousto that reported for the polyribosyl ribitol phosphate-outer membraneprotein complex from Neisseria meningitiidis (Mach et al., Biotechniques15:240-242, 1993). Under all storage conditions, this peak had a higherabsorption at 254 nm, compared to that at 280 nm, as evaluated from theHPLC traces and peak integration. The immunoreactivity of the fourdifferent forms with MPA3 and MAB71 were determined (FIG. 22A). Thehexamer and octomer forms of recombinant urease showed apparentlysimilar reactivities with MPA3 and MAB71. The polymeric form (P) showedmuch lower reactivity with both MPA3 and MAB71.

Electron micrograph of different forms recombinant urease

Electron microscopic evaluation (FIG. 22B) of octomeric (0) andhexameric (H) forms revealed that these fractions are composed ofrelatively pure monodispersed macromolecular particles that are 12 nm indiameter, and most of which are oriented with the central core facingup. The high molecular weight, polymeric (P) form that eluted in thevoid volume of the column consisted of a mixture of 12 nm macromolecularparticles and randomly oriented particulate material aggregated togetherinto thick masses that failed to disperse over the carbon surface. Thetetrameric form (T) of urease consisted of finely dispersed particles ofless than 10 μm and a few isolated 12 nm particles.

Effect of β-mercaptoethanol, EDTA, and Tween 20 on the stabilityofrecombinant urease The effect of β-mercaptoethanol, EDTA, and Tween 20on the stability of purified recombinant urease was also studied. Table2 shows the effect of β-mercaptoethanol on the stability of purifiedrecombinant urease, as evaluated by HPLC analysis. Dialysis of samplesagainst PBS for 12-24 hours resulted in formation of high molecularweight forms of urease. This was evident from detection of a significantreduction in the total area of the urease peak analyzed immediatelyafter dialysis (peak area 51), compared to the urease dialyzed in PBScontaining 0.01-1 mM urease (peak area 72-80). Protein analysis by amodified Lowry method (Zak et al., Clin. Chim. Acta 6:665-670, 1961) andSDS-PAGE under reducing conditions revealed no difference in proteinconcentration or UreB and UreA band intensities between the samplesdialyzed in PBS in the absence or presence of β-mercaptoethanol. Theseresults show that the urease dialyzed in PBS is aggregated and isremoved by the Z-spin filters used in filtering the samples for HPLCanalysis. Formation of high molecular weight forms of urease upondialysis against PBS is also supported by detection of a significantincrease in the area of the high molecular weight urease peak eluting atthe void volume. Inclusion of 0.01-1 mM β-mercaptoethanol in thedialysis buffer prevented the molecular associations. EDTA (1 mM) orTween 80 (0.2%) did not have any significant effect on this. The extentof association and precipitation continued to increase with time ofstorage in the absence of β-mercaptoethanol. During 1 week of storage at4° C., in the absence of β-mercaptoethanol, nearly 90% of urease wasremoved by filtration using the 0.2 μm Z-spin filter, as evident fromthe reduction in the total area (peak area 51.0 in day 1 and 5.0 in day7). β-mercaptoethanol (0.01 mM to 1 mM) afforded significant protectionagainst this molecular association. The presence of β-mercaptoethanolnot only prevented the association of urease into different, highmolecular weight forms, but it also resulted in the breakdown of some ofthe urease to small molecular weight components. Sucrose significantlyreduced the extent of aggregation of urease, and, in the presence ofsucrose, the effect of β-mercaptoethanol was marginal. The protectiveeffect of β-mercaptoethanol can be explained by the ability ofβ-mercaptoethanol to prevent inter-subunit disulfide bridges betweenUreA and UreB. The increased breakdown of the high molecular weightforms to lower molecular weight forms can similarly be explained by theability of β-mercaptoethanol to break intermolecular disulfide bondsbetween urease subunits.

Effect of DTNB treatment on the molecular state and immunochemicalproperties of recombinant urease

These results show stabilization of recombinant urease byβ-mercaptoethanol. Because instability caused by molecular associationleads to inter-subunit disulfide bridges, blocking of free SH groups ofurease should prevent the molecular association. To test this, DTNB wasused to block the free sulfhydryl groups in the recombinant protein. Theamino acid composition predicted by the nucleic acid sequences of thegenes encoding UreA and UreB suggest the presence of three cysteineresidues in each UreB subunit and one SH group in each UreA subunit.Thus, the nucleotide sequences of the genes encoding UreA and UreBpredict the presence of four SH groups for a urease (UreA-UreB)monomeric unit. The SH groups of recombinant urease were titrated withDTNB (FIG. 23A). More than three moles of SH groups were titrated foreach mole of (UreB-UreA) monomer of freshly prepared urease. Thisobservation shows that 70-80% of the total SH groups are free andtitratable with DTNB. Addition of 1% SDS to the reaction mixtureresulted in enhanced reactivity of the SH groups, but the total numberof SH groups titrated was the same in the presence and absence of SDS.Non-reducing SDS-PAGE analysis of a freshly prepared urease sample,followed by Coomassie staining and densitometry, revealed that 70-75% ofthe UreB and UreA subunits are non-covalently associated and breakdownin SDS. The remaining 20-30% were covalently associated and migrated ashigh molecular weight protein bands above the UreB band. This resultconfirms the presence of inter-subunit disulfide linkages in recombinanturease. The results from non-reducing SDS-PAGE were consistent with theDTNB titration results.

SH group reactivity decreases with time of storage

Recombinant urease solutions stored at 4° C. for different time periodsshowed a time-dependent reduction in titratable SH groups (FIG. 23B).Consistent with this reduction in free SH groups, there was an increasein high molecular weight protein bands as detected by non-reducingSDS-PAGE (FIG. 24B, lanes 1-3) and native PAGE (FIG. 24C, lanes 1-3),with very little change detected by reducing SDS-PAGE (FIG. 25A, lanes1-3). The high molecular weight protein bands detected by non-reducingSDS-PAGE reacted with MPA3 (FIG. 25A), MPA4 (FIG. 25B), and MPA6 (FIG.25C), suggesting the formation of UreA-UreB covalent associations.

Blocking the SH groups stabilizes recombinant urease by preventingmolecular associations

To determine the role of sulfhydryl groups on the molecular associationsof urease, the free SH groups were blocked by titration with DTNB. Theproperties of DTNB-treated urease were compared with those of untreatedcontrols (FIGS. 24 and 25). An analytical size exclusion HPLC profile ofDTNB-treated urease is compared with those of untreated controls in FIG.23C. Dialysis of urease in PBS under the experimental conditionsresulted in a significant reduction in the area of the hexameric andoctomeric urease, with a concomitant increase in the high molecularweight urease peak in the column void volume (FIG. 23C, chromatogram c).However, DTNB treatment, followed by dialysis in PBS, prevented themolecular associations (FIG. 23C, chromatogram b). In fact, theDTNB-treated sample showed a slight increase in the low molecular weighturease peak (tetrameric or pentameric urease). These results (FIGS.24A-24C, lanes 5 and 6, FIGS. 25A-25C, lanes 3 and 4, and FIG. 23C) showthat DTNB treatment stabilized the hexamer form of urease and preventedmolecular association. The control urease, not treated with DTNB, butotherwise treated identically to the DTNB-treated samples, was highlypolymerized. DTNB treatment did not affect the immunoreactivity of theurease, as determined by Western Blot analysis. DTNB-treated samplesretained immunoreactivity with MPA3 and MAB71, as determined by ELISAanalysis, but the control sample, which was treated identically, hadmuch lower ELISA reactivity. Alkylation of recombinant urease usingiodoacetamide also stabilized the hexameric form of urease and preventedmolecular association.

Freshly isolated urease stored in 20 mM Tris pH 8.6 was alkylated byincubation with 1 mM iodoacetamide for 1 hour at room temperature. Theresulting product was diafiltered into 2% sucrose. The alkylated andcontrol urease were titrated with DTNB and the number of titratable SHgroups for each monomeric urease was estimated. DTNB titration offreshly isolated urease showed the presence of 2.5-3.0 moles oftitratable SH groups and analysis of the alkylated urease showed thepresence of 0.4 titratable SH groups in the absence of SDS. In thepresence of 1% SDS, the alkylated urease showed the presence of 1-1.2moles of SH group and control urease showed the presence of nearly 3.1moles of SH groups. These results showed that nearly three SH groups arefree for every UreA-UreB complex, and, under the experimentalconditions, two of the SH groups are blocked by iodoacetamide. The freeSH group that was not blocked by iodoacetamide became reactive with DTNBin the presence of 1% SDS, but not in the absence of SDS.

The abbreviations used in the description set forth above are asfollows: HPLC, high pressure liquid chromatography; FPLC, fast proteinliquid chromatography; MPA, mouse polyclonal ascites; PBS,phosphate-buffered saline (10 mM Phosphate, 150 mM sodium chloride, pH7.4); DTNB, 5-5'-Dithiobis-(2-Nitrobenzoic acid); PEM, 20 mM phosphate,1 M EDTA, 1 mM β-mercaptoethanol; and CAPS,3-[cyclohexylamino]-1-propanesulfonic acid.

                  TABLE 1                                                         ______________________________________                                        Amino acid composition of recombinant H. pylori urease compared with           the amino acid compositions of native H. pylori urease and                    ureases from other sources.                                                        Mole % in different ureases.sup.c                                                                             Brevibac-                                    Jack K. tetium                                                             Amino  H. pylori Bean aerogenes ammonia-                                      Acid.sup.a r. urease.sup.b urease.sup.d urease.sup.e urease.sup.f genes                                           urease.sup.g                            ______________________________________                                        Asx*  11.8(0.4) 15.6     10.6  9.2    11.6                                      Glx** 11.1(1.2) 11.2 8.2 10.3 10.2                                            Ser 4.5(0.1) 5.1 5.6 6.1 3.6                                                  Gly 9.5(0.3) 11.1 9.4 12.4 9.7                                                His 3.4(0.2) 5.6 3.0 3.0 2.9                                                  Arg 3.2(0.8)*** 3.9 4.5 4.0 4.9                                               Thr 7.2(0.4) 6.2 6.6 6.9 7.2                                                  Ala 9.4(0.5) 9.8 8.6 10.5 10.7                                                Pro 4.1(0.1) 3.4 5.0 5.5 4.7                                                  Tyr 2.3(0.2) 2.1 2.5 6.9 7.2                                                  Val 5.8(0.2) 6.1 6.6 7.5 7.8                                                  Met 3.4(0.4) 2.4 2.5 1.7 1.8                                                  Cys                                                                           Ile 6.9(0.3) 5.9 7.9 5.7 7.1                                                  Leu 6.4(0.3) 7.4 8.2 6.6 7.5                                                  Phe 3.8(0.5) 4.1 2.9 2.6 3.0                                                  Lys 7.3(0.5)                                                                  Try                                                                         ______________________________________                                         .sup.a Cys, Lys, and Try were not detectable by the methods used.             .sup.b Average of 7 estimations with standard deviations in parentheses.      .sup.c Amino acid composition predicted from the nucleotide sequence.         .sup.d Data from Hu et al., Infection and Immun. 58:992-998, 1990.            .sup.e Data from Mamiya et al., Proc. Jpn. Acad. 61:395-398.                  .sup.f Data from Todd et al., J. Biol. Chem. 263:5963-5967, 1987.             .sup.g Data from Nakano et al., Agric. Biol. Chem. 48:1495-1502, 1984.        *Represents sum of asparagine and aspartic acid.                              **Represents sum of glutamine and glutamic acid.                              ***Very high standard deviation, between the results from runs at two         different time points.                                                   

                                      TABLE 2                                     __________________________________________________________________________    Effect of β-mercaptoethanol on the stability of urease                   Condition                                                                              Peak    Peak    Peak area                                                                             Total urease                                   β-mercaptoethanol area (Hexamer) area (Octomer) Higher form peak                                        area                                         (mM)     Day 1                                                                             Day 7                                                                             Day 1                                                                             Day 7                                                                             Day 1                                                                             Day 7                                                                             Day 1                                                                             Day 7                                    __________________________________________________________________________    0.0      39.1                                                                              3.2 9.3 1.1 2.13                                                                              0.66                                                                              51.0                                                                              5.0                                        0.01 60.2 30.04 9.1 7.64 2.7 1.64 72.0 39.3                                   0.1 73.2 54.7 3.4 3.3 3.2 2.0 80 60                                           1.0 75 49 0.0 0.0 3.2 1.1 78 50.1                                           __________________________________________________________________________     Purified recombinant urease (2-2.3 mg/ml) was dialyzed for 24 hours           against phosphatebuffered saline, pH 7.0, containing 0, 0.01, 0.1, and 1      mM mercaptoethanol. The samples were analyzed immediately after dialysis      and 7 days after storage at 4° C. by analytical size exclusion         HPLC. Samples were prefiltered using 0.2 μm Zspin filters before           injection into HPLC columns. The absolute peak area of different forms of     urease is shown. The hexamer form(Rt = 9.2-9.4 minutes),  # octomer form      (Rt = 8.2-8.4 minutes), and higher molecular weight form (Rt = 6.0            minutes) are evaluated.                                                  

The results set forth above were obtained using the following materialsand methods.

Bacterial strains and media

H. pylori strain ATCC 43504 (American Type Culture Collection,Rockville, Md.) was used for the production and purification of ureasefor the generation of immunological reagents. H. pylori strain CPM 630(from Soad Tabaqchali's Laboratory, St. Bartholomew's Hospital MedicalSchool, London) was used for production and purification of native H.pylori holoenzyme for comparison with recombinant urease. H. pylori wasgrown in Mueller Hinton (MH) agar (Difco Laboratories, Detroit, Mich.)containing 5% sheep red blood cells (Crane Labs, Syracuse, N.Y.) andantibiotics (5 μg/ml trimethoprim, 10 μg/ml vancomycin, and 10 Upolymyxin B sulfate (TVP) per ml; Sigma Chemical Co., St. Louis, Mo.).Plates were incubated for 3-4 days at 37° C. in 7% CO₂ and 90% humidity.

Production and purification of urease

The expression, production, and purification of recombinant urease, aswell as the purification of native H. pylori urease and generation ofantibodies, has been described (Lee et al., J. Infect. Dis. 172:161-172,1995; Pappo et aL, Infect. Inuun. 63. 1246-1252, 1995). Recombinant H.pylori urease was produced in genetically engineered E. coli transformedwith a plasmid containing the structural genes of H. pylori urease. Theproduction strain, designated ORV214, contained the structural genesencoding UreA and UreB in an isopropylthiogalactoside (IPTG)-inducibleexpression system (Lee et al., J. Infect. Dis. 172:161-172, 1995; Pappoet al., Infect. Immun. 63:1246-1252, 1995).

Recombinant urease was produced in large scale by growing therecombinant E. coli strain (ORV214) in 40-400 liter fermentation tanks.Bacteria from the fermentation systems were harvested by centrifugation.Urease was extracted by breaking the bacteria in 20 mM phosphate, 1 mMEDTA (PE) buffer, pH 6.8, by passing the bacteria through aMicrofluidizer at a pressure of 13,000-19,000 psi, and clarified bycentrifugation at 28,000-30,000× g in a Sorvall 6C high speed centrifugeat 4° C. The soluble urease from the extract was purified using acombination of ion-exchange chromatography on DEAE-Sepharose andQ-Sepharose (Pharmacia Biotechnology), and by an ultrafiltrationdiafiltration procedure.

Native H. pylori urease was purified using a modification of theprocedure reported by Hu et al. (Infect. Immun. 58:992-998, 1990). H.pylori strain ATCC 43504 or CPM 630 was grown in blood agar plus TVPAplates. Bacteria were harvested by centrifugation (20,000× g in Sorvall6C high speed centrifuge for 15 minutes at 4° C.), resuspended in 3volumes of water or 20 mM phosphate, 1 mM EDTA, 1 mM β-mercaptoethanol(pH 6.8), lysed by sonication using a Branson sonifier (Ultrasonics,Danbury, Conn.) with three 15 second pulses, at 50% duty cycle and apower setting of and clarified by centrifugation (30,000× g for 45minutes at 4° C.). The clarified supernatant was mixed with 3 M sodiumchloride to a final sodium chloride concentration of 0.15 M and passedthrough a 1.6×10 cm DEAE-Sepharose column (Fast Flow). The fractionswith urease activity that passed through the column under theseconditions were collected, concentrated using a Filtron Macrosep 100centrifugal filtration unit, and then passed through a Superose 12 (1×30cm) or Superdex 200 (1.6×60 cm) size exclusion column. Superose 12chromatography was performed at a sample load of 0.5 ml and a flow rateof 30 ml/hour. Superdex-200 column chromatography was performed at asample load of 2-4 ml and a flow rate of 120 ml/hour. Size-exclusionchromatography was performed using Pharmacia prepacked columns and anFPLC system. The fractions containing urease activity were pooled,concentrated, and further purified by anion-exchange chromatography on aMono Q-Sepharose column. Mono Q anion-exchange chromatography wasperformed using a prepacked (0.5×5 cm) column from Pharmacia and an FPLCsystem. The column was equilibrated with PEM buffer. The bound ureasewas eluted using a 0-1 M sodium chloride gradient in PEM buffer. Thefractions with urease activity were pooled and concentrated usingMacrosep 100 centrifugal filters. In cases where the purity was lessthan 90%, as determined by SDS-PAGE and densitometry, furtherpurification was achieved by a final analytical size exclusion FPLCfractionation on Superose 12 columns.

Production of high titer anti-urease polyclonal mouse antibodies

Polyclonal mouse antibodies were raised against the purified H. pyloriurease and urease subunits. The MPA3 antibody was generated usingpurified urease. The UreB and UreA subunits were purified by separatingthe subunits by SDS-PAGE and electroeluting the separated subunits fromgel slices. The electroeluted proteins were used for generation of MPA4and MPA6. The generation of MPA3 (anti-urease), MPA4 (anti-UreA), andMPA6 (anti-UreB) have been described (Lee et al., J. Infect. Dis.172:161-172, 1995). A rabbit polyclonal antibody against purified H.pylori urease was generated by subcutaneous injection of purified urease(150 μg) in Freund's complete adjuvant, followed by 2 booster doses ondays 27 and 45 with 150 μg purified urease in Freund's incompleteadjuvant. Serum IgG was purified by ammonium sulphate precipitation anddialysis (Lee et al., J. Infect. Dis. 172:161-172, 1995). A monoclonalhybridoma that secretes IgA recognizing the UreB subunit was produced inthe laboratory of Dr. S. Czinn (Blanchard et al., Infect. Immun.63:1394-1395, 1995). This hybridoma was prepared using H. felis sonicateantigens. The hybridoma was grown in serum-free and protein-free medium(Sigma).

Analytical Methods

Analytical columns (Superdex 200, Superdex 75, and Superose 12 columns)were either obtained as prepacked FPLC columns or packed in-house usingPharmacia XK series columns with adaptors and resins from Pharmacia-LKB.Analytical size exclusion HPLC columns (Progel TSK-G4000 SW×L, 7.8 mm×30cm) and Progel SW×L guard columns were obtained from SupelCo. Thechromatographic resins DEAE-Sepharose (FF) and Q-Sepharose (FF) wereobtained from Pharmacia.

Analytical size exclusion HPLC of purified urease was performed usingthe Gold HPLC system from Beckman, Inc. The system consisted of Pump126, Diode array UV-visible dual wavelength detector 168, and SystemGold software package V711. Chromatography was performed under isocraticconditions using a Progel TSK 4000 SW×L (7 μm) column (7.8 mm×30 cmi.d.) and a GW×L guard column in 100 mM phosphate, 100 mM sodiumchloride (pH 7.0) at a flow rate of 1 ml/minute, and a pressure of450-500 psi.

Reducing SDS-PAGE was performed in 10% or 12.5% polyacrylamide gels.Non-reducing SDS-PAGE was performed using a 4-20% precast gradient gelfrom NOVEX (Novel Experimental Technology, San Diego, Calif.).Native-PAGE was performed using a 4% or 6% polyacrylamide gel.Densitometric scanning of Coomassie stained gels was performed using theUltroscan XL laser densitometer from Pharmacia-LKB.

For Western blots, proteins separated on polyacrylamide gels weretransferred to nitrocellulose paper, blocked with 2.5% nonfat dry milkin 50 mM Tris, 0.5 M NaCl (pH 7.5), and then incubated overnight at 4°C. with anti-urease antibodies appropriately diluted in the blockingbuffer. The unbound antibodies were washed off by three minute washingswith blocking buffer. The blotted papers were then incubated withalkaline-phosphatase conjugated anti-mouse IgG for 2 hours at roomtemperature. The unbound antibodies were washed off by three 10 minutewashings with 50 mM Tris, 0.5 M NaCl and then developed using Sigmafastalkaline-phosphatase substrate (1 tablet dissolved in 10 ml water).

For N-terminal sequencing, the UreA and UreB subunits were separated by10% reducing SDS-PAGE, transferred to a transblot PVDF membrane (BioRad)using 10 mM CAPS, 10% methanol, pH 11.0, stained using 0.1% Coomassiebrilliant blue R in 50% methanol, destained using 50% methanol, washed,and air-dried. N-terminal sequencing was performed at the MolecularBiology Core facility at Dana Farber Cancer Institute, Boston, Mass.Sequencing was performed on an Applied Biosystem automated sequencerusing post-liquid Technology by Edman degradation.

For analysis of total amino acid composition, the purified product wasextensively dialyzed against HPLC-quality water. Aliquots of dialyzedsamples were immediately transferred to hydrolysis tubes. Amino acidhydrolysis and analysis was performed at the Molecular Biology Corefacility at Dana-Farber Cancer Institute, Boston, Mass. Hydrolysis wasperformed with 6 N HCl in vacua containing phenol for 1 hour at 110° C.and analysis was performed using an analyzer from ABI and the Pico-tagmethod.

For characterization of different molecular forms of urease formedduring the course of storage under different conditions, the molecularforms were separated by analytical size exclusion HPLC. Differentfractions were collected and analyzed by native and reducing SDS-PAGE,Coomassie blue staining, and Western blot analysis using anti-ureaseantibodies. The fractions corresponding to the peak positions ofdifferent molecular forms were analyzed for ELISA reactivity using themouse polyclonal anti-urease antibody MPA3 and mouse monoclonalanti-urease IgA MAB71 in a standardized ELISA. ELISA reactivity usingthese antibodies was measured using a standardized urease capture ELISA(Lee et al., J. Infect. Dis. 172:161-172, 1995).

The HPLC-separated urease peaks were applied to carbon support filmcopper grids (Electron Microscopy Sciences, Fort Washington, Pa.),negative stained with 1% ammonium molybdate containing 0. 1% glycerol,pH 7.5, and examined with a JEM-1010 electron microscope (JEOL, Inc.,Peabody, Mass.). The original magnification was 240,000×.

The total number of reactive thiol groups in urease was estimated byspectrophotometric titration at 412 nm using 5-5'-Dithio-bis-(2-Nitrobenzoic acid) (DTNB). DTNB-blocked urease was prepared by incubatingpurified, reconstituted urease in 100 mM Tris-HCl (pH 8.0) with 0.4%DTNB. The total number of SH groups reacted were calculated using amolar extinction coefficient of 13,000 (Ellman, Arch. Biochem. Biophys.74:443-450, 1958), as determined using a standard curve run undersimilar conditions using cysteine.

Stabilization of Urease by Genetic Modification

We have constructed several urease structural protein mutants in whichfunctional residues were substituted with alanine or other nonfunctionalresidues. These mutants include, for example, strain ORV261, in whichhistidine 136 is substituted with alanine (H136A), and ORV273, in whichlysine 219 is substituted with alanine (K219A). ORV273 also includes ahistidine 248 to alanine (H248A) substitution, which was introduced bychance (FIG. 26D). Construction and testing of these nonactivatablemutant forms of recombinant apourease is described further, as follows.

UreA and UreB genes were obtained from Institute Pasteur's clone pILL944(pET11a (ureA+B)). This plasmid contains an engineered NdeI site at thestart codon for ureA, which minimizes the untranslated region betweenvector regulatory sequences and the start of ureA. The NdeI/EcoRIfragment of pILL944, containing the ureA and ureB units, was subclonedinto similarly digested pET29a+to create pORV261. The plasmid wasintroduced into BL21 DE3 for expression of UreA and UreB under T7regulatory control. This strain (ORV261) was confirmed to express ureaseand the strain could be cultivated and urease purified from it usingmethods for ORV214. The nucleotide sequence for ureA and ureB wasconfirmed to match the published sequence. This plasmid was used astemplate for introduction of subsequent mutations intended forprevention of reactivation.

Specific amino acid substitutions were introduced into pORV261 via sitedirected mutagenesis using the Kunkel method (Kunkel et al., Methods inEnzymology 154:367, 1987; Kunkel, Proc. Natl. Acad. Sci. USA 82:488,1985). We have constructed several mutant strains. Two of the mutantstrains (ORV261-H136A and ORV261-K219A plus H248A) were cloned andcharacterized extensively. Recombinant E. coli strains expressing H136Amutant urease and K219A plus H248A double mutated urease structuralgenes produced urease structurally analogues to the apourease producedby ORV214. Urease purified from E. coli pellets were purified by similarmethod used for purification of apourease from ORV214 pellet. Ureasepurified from the mutant strains were structurally identical to theurease purified from ORV214 strain, as detected by analytical sizeexclusion HPLC, SDS-PAGE, and Coomassie staining, and did not exhibiturease enzymatic activity. Unlike the apourease from ORV214, theapourease from this mutant strain was not activated in vitro byincubation with bicarbonate and nickel ions. This data confirmed thatmutations H136A and the double mutation K219A plus H248A resulted inprevention of in vitro activation.

The K219A plus H248A double mutant was further characterized. Thismutation was confirmed in clone 273 by sequencing of mutation and falllength sequencing of the urease insert. (The attempted mutation wasK219A and the H248A mutation was incidental.) Research cell bank andmaster and working cell banks are constructed for production of urease.Urease was purified from fermentation pellets of ORV273 working cellbanks using a method similar to that used for purification ORV214pellet. The purified urease was structurally similar to apoureaseproduced from ORV214, was not enzymatically active, and was notactivatable in vitro under conditions in which apourease from ORV214 isactivated (FIGS. 26A-26D).

The apourease purified from ORV273 was tested in a mouse model for itsefficacy in protecting against H. pylori challenge. In a comparativestudy using urease from ORV273 and ORV214, both induced similar immuneresponses and showed equal efficacies in protecting the mice frombacterial infection (FIG. 27). This observation confirms that blockingin vitro activation has no effect on the protective effect of ureaseagainst Helicobacter infection. In addition, genetically altered ureasepurified from strain ORV273 showed similar reactivity as wild typeurease with MAB71, which is a monoclonal IgA antibody raised againstUreB (Table 3). These results further show that the mutations in ORV273do not affect the antigenicity, immunogenicity, and protective effect ofurease against bacterial challenge.

                  TABLE 3                                                         ______________________________________                                        Comparison of the immunoreactivity of recombinant mutant urease                 purified from strain ORV273 with the immunoreactivity of recombinant         urease purified from strain ORV273                                                          Relative ELISA titer to                                                                       Relative ELISA titer to                          Recombinant Urease MAb71* MPA3*                                             ______________________________________                                        ORV214      92%            102%                                                 ORV273 122% 111%                                                            ______________________________________                                         *ELISA titer is calculated from standard curves constructed using             reference standard purified urease from strain ORV214. The reactivity of      the urease used in standard curve is arbitrarily taken as 100% for            comparison. The reactivity of urease from strain ORV273 is similar to tha     of the standard recombinant urease from ORV214, within the assay              variability. ELISA titrations were performed using standardized urease        capture ELISA, as described in analytical methods, and the values were        calculated using  # standard protocols used in the laboratory.           

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Glu Ala Gly Ala Ile Gly Phe Ala Ile His - #Glu Asp Trp Gly Thr           1               5 - #                 10 - #                 15              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  GAAGCCGGTG CGATTGGCTT TGCAATTCAC GAAGACTGGG GCACC  - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Gln Val Ala Ile Ala Thr Asp Thr Leu                                       1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  CAAGTCGCTA TCGCCACAGA CACTTTG         - #                  - #                 27                                                                    __________________________________________________________________________

What is claimed is:
 1. A method of preventing activation of inactive,recombinant Helicobacter pylori apourease comprising reacting saidapourease with a compound that modifies a sulfhydryl or lysyl residue ofsaid apourease.
 2. The method of claim 1, wherein said amino acidcomprises a sulfhydryl group and said compound is a sulfhydryl-reactivecompound.
 3. The method of claim 2 wherein said compound isiodoacetamide (IAM) or iodoacefic acid (IAA).
 4. The method of claim 2,wherein said compound is selected from the group consisting of5-5'-dithio-bis-2-nitrobenzoic acid (DTNB), 2,2'-dithiodipyridine(DTDP), cystine, cystamine, methyl methanethiolsulfonate (MMTS), N-ethylmaleimide (NEM), dinitrofluorobenzene (DNFB), and trinitrobenzenesulfonic acid (TNBSA).